fusion protein, polynucleotide, construct, vector, host cell, pharmaceutical composition or vaccine,

专利摘要:
FUSION PROTEIN, POLYNUCLEOTIDE, CONSTRUCT, VECTOR, HOST CELL OR ORGANISM, PHARMACEUTICAL COMPOSITION OR VACCINE, USE OF THE FUSION PROTEIN, METHOD TO PREVENT OR TREAT HEV INFECTION AND THE TREATMENT OF HEALTHCARE INFECTED HEALTHCARE INFECTED COMBINATION DISEASES ASSOCIATED WITH INFLUENZA VIRUS INFECTION, METHOD FOR IMPROVING THE IMMUNOGENICITY OF A TARGET PROTEIN AND THE USE OF CRM197 OR A FRAGMENT OF THE SAME. A CRM197 mutant non-toxic diphtheria toxin or a fragment thereof as an adjuvant in an adjuvant to a fusion protein is provided in the present invention and the use thereof to improve the immunogenicity of a target protein fused thereto, for example , a HEV capsid protein, or an M2 protein from the influenza virus or an immunogenic fragment thereof. Also provided is a method for improving the immunogenicity of a target protein, comprising expressing the CRM197 protein or fragment thereof with the target protein to form a fusion protein. Additionally provided is a fusion protein comprising the CRM197 or its fragment and a target protein, the CRM197 or its fragment improves the immunogenicity of the protein (...).
公开号:BR112013030963B1
申请号:R112013030963-6
申请日:2012-06-01
公开日:2020-12-01
发明作者:Shaowei Li；Cuiling Song；Chunyan Yang；Ying Gu；Wenxin Luo；Ningshao Xia
申请人:Xiamen University；Xiamen Innovax Biotech Co., Ltd.；
IPC主号:

专利说明:

Field of invention
[001] The invention relates to a field of molecular virology and immunology. In particular, the invention relates to the use of a non-toxic diphtheria toxin CRM197 or a fragment thereof as an intramolecular adjuvant in a fusion protein to improve the immunogenicity of a target protein fused to it (for example, a HEV capsid, an M2 protein from the influenza virus or an immunogenic fragment thereof). The invention also relates to a method for improving the immunogenicity of a target protein (for example, a HEV capsid protein, an M2 protein from the influenza virus or an immunogenic fragment thereof), comprising the expression of the CRM197 fusion or a fragment of it with a target protein to form a fusion protein. The invention also relates to a fusion protein comprising CRM197 or a fragment thereof and a target protein (for example, a HEV capsid protein, an M2 protein from the influenza virus or an immunogenic fragment thereof), said CRM197 being o a fragment of it improves the immunogenicity of the target protein. The invention also relates to an isolated nucleic acid encoding the fusion protein, a construct and a vector comprising the nucleic acid, and a host cell comprising the nucleic acid. The invention also relates to the use of the fusion protein in the manufacture and a pharmaceutical composition or vaccine. Background of the invention
[002] Diphtheria toxin (DT) has been extensively studied. Structure studies demonstrate that diphtheria toxin consists of three domains: catalytic N-terminal C domain (aa 1-190, domain C) (also called fragment A), intermediate transmembrane T domain (aa 201-384, domain T), and the C-terminal receptor-binding R domain (aa 386-535, R domain) (Choe s, Bennett M., Fujii G, et al., “Nature”, 1992, 357: 216-222 ). ONTAK (DAB389-IL-2), prepared by the merger of two domains of diphtheria toxin with interleukin 2 (IL-2), was approved by the FDA on the market in 1999 for the treatment of cutaneous T-cell lymphoma in adults. This demonstrates that the three domains of diphtheria toxin can be used separately and represent their own rules, respectively.
[003] CRM197 (cross reaction material 197) is a non-toxic mutant diphtheria toxin (Uchida, T., AM, Pappenheimer, Jr., R. Gregory, et al., “J. Biol. Chem.”, 1973, 248: 3838-3844), which differs from an allele-wild gene encoding DT by a single nucleotide mutation, resulting in the amino acid residue at position 52 changed from Gli to glu (G. Giannini, R. Rappuoli, G. Ratti et a., "Nucleic Acids Research", 1984, 12: 4063-4070).
[004] Studies show that although CRM197 has a structure similar to that of allele-wild DT (called, having the said three domains), its Fragment A loses its ability to bind to NAD ,, it is unable to bind to EF2 and thus loses the cytotoxicity possessed by natural DT, indicating that the amino acid residue Gli, at position 52, participates in an important rule in the connection of DT to NAD (K. Moyner, G. Christiansen, “Acta Path Microbial Immunolscand Sect C ”, 1984; 92: 17-23). Although CRM197 loses cytotoxicity, it retains a strong immunogenicity comparable to that of an allele-wild DT. Therefore, CRM197 is generally used as a protein carrier for crosslinking other haptens in order to prepare conjugate vaccines.
[005] As previously in 1985, Porter et al., Cross-linked the polysaccharides on the Hib surfaces to CRM197 and the DT protein vehicle, respectively, and prepared them in vaccines, and studied their difference in immunogenicity. The results of the experiments demonstrated that there is no significant difference between the two cross-linked vaccines in terms of immune effect, both of which could stimulate the production of a strong immune response and immune memory in children (Porter Anderson, Micheal E. Pichichero and Richard A., “ J. clin. Invest. ”, 1985: 5259). After comparing cross-linked pneumococcal conjugate vaccines to various proteins, it was observed that vaccines, where CRM197 was used as a protein carrier, had a good immune effect in animal experiments and clinical screenings, and CRM197 is safe without a side effect of toxicity ( Black, s., H. Shinefield, et al., "Pediatr. Infect. Dis. J.", 2000, 19 (3); 187-195). Currently, pneumococcal conjugate vaccines, in which CRM197 is used as a protein carrier, refer mainly to PCV7, PCV9, PCV13, and the like. The results of clinical screenings demonstrated that these vaccines had good immunogenicity and safety in children under two years of age (Barricarte, A., “J. Castilla, et al.,“ Clin. Infect. Dis. ”, 2007, 25 (13): 2451- 2457; Duggan, ST Drugs, 2010, 70 (15): 1973-1986). Epidemic meningitis conjugate vaccines can be prepared by cross-linking CRM197 to polysaccharides on the surface of N. menigitidis. For example, vaccines such as Meningitec (Wyeth Pharmaceutcals), Menjugate (Novartis vaccines), and Menveo (Novartis vaccines), in which CRM197 was used, as a protein carrier, are commercially available.
[006] Although CRM197 loses enzymatic activity and cytotoxicity, it is still able to bind to a specific Dt receptor, that is, heparin-binding EGF-like growth factor (HB-EGF). Since receptor expression is generally over-regulated in cancerous tissues, type DT, CRM197 also has an anti-tumor effect (Buzzi, S., D. Rubboli, et al., “Immunotherapy”, 2004, 53 ( 11)). Studies have also observed that CRM197 could pass through the brain-blood barrier (BBB) and therefore could be used as a vehicle to deliver drugs to the brain (Gaillard, PJ, and Boer AG, “J. Control Release”, 2006, 116 (2): 60-62).
[007] Although it has been reported that CRM197 has multiple functions, in particular, it had a strong immunogenicity and can be used as an immunoadjuvant it has been reported that CRM197 can be used as an intramolecular adjuvant to improve the immunogenicity of a target protein fused to it into a fusion protein. The invention uses the hepatitis E capsid protein as an example, and demonstrates in the first instance that CRM197 or a fragment thereof can improve the immunogenicity of a protein fused to it in a fusion protein and, therefore, can be used as an intramolecular adjuvant. Description of the invention
[008] In the invention, unless otherwise specified, the technical and scientific term used here has the meaning generally understood by a person skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, biological chemistry, and immunological laboratory operations used here are routine operations widely used in corresponding fields. For the time being, in order to better understand the invention, the definitions and explanations of relevant terms are provided below.
[009] According to the invention, the term "CRM197" refers to a mutant non-toxic diphtheria toxin, which differs from an allele-wild diphtheria toxin by an amino acid residue at the 52 position changed from Gli to Glu (G. Giannini, R. Rappuoli, G. Ratti et al., “Nucleic Acids Research”, 1984, 12: 4063-4070). Diphtheria toxin is well known to those skilled in the art (see, for example, Choe S., Bennett M., Fujii G., et al., “Nature”, 1992, 357: 216-222), the sequence of which can be observed with reference to the access number in GenBank No. AAV70486.1.
[010] In the invention, the exemplary amino acid sequence of CRM197 is represented in SEQ ID NO: 2. Therefore, in the invention, when the CRM197 sequence is involved, it was described as the sequence represented in SEQ ID NO: 2. For example, in the expression "amino acid residues of positions 1 to 190 of CRM197", the amino acid residues of positions 1 to 190 refer to amino acid residues from positions 1 to 190 of SEQ ID NO: 2. However, a person skilled in the art understands that mutations or variations (including, but not limited to, substitution, deletion and / or addition) can naturally occur in or are artificially introduced into SEQ ID NO: 2 without affecting the biological properties of CRM197. Therefore, in the invention, the term "CRM197" is intended to comprise all of said polypeptides and variants, including the polypeptide represented in SEQ ID NO: 2 and its natural or artificial variants, with the variants retaining the biological properties of CRM197; that is, it has a strong immunogenicity and no cytotoxicity, they include not only fragments of the sequence of a polypeptide represented in SEQ ID NO: 2, but also fragments of the corresponding sequence of the natural or artificial variants of the polypeptide. For example, the term "amino acid residues from positions 1 to 190 of CRM197" is intended to comprise amino acid residues from positions 1 to 190 of SEQ ID NO: 2 and the corresponding fragments of the variants (natural or artificial) of a polypeptide represented in SEQ ID NO: 2.
[011] According to the invention, a Hepatitis virus (HEV) capsid E protein refers to a protein encoded by HEV ORF2. The sequence of HEV ORF2 is well known in the state of the art (see, for example, the accession number in DDBJ No .: D11092). In the invention, when the HEV ORF2 sequence is involved, it is described as the sequence represented by the accession number in DDBJ No .: D11092. For example, in the expression “amino acid residues of positions 368 to 606 of a polypeptide encoded by HEV ORF2”, amino acid residues of positions 386 to 606 refer to amino acid residues of positions 368 to 606 of a polypeptide encoded by D11092. However, a person skilled in the art understands that mutations or variations (including, but not limited to, substitution, deletion and / or addition) can naturally occur in or are artificially introduced into the HEV ORF2 or an encoded polypeptide without affecting its biological properties. (such as antigenicity and immunogenicity). However, in the invention, the term "HEV ORF2" is intended to comprise all said polypeptides and variants, including the sequence represented in D11092 and its natural or artificial variants. In addition, when HEV ORF2 sequence fragments (or a polypeptide encoded by the same) are described, they include not only fragments of the D11092 sequence (or at least one polypeptide encoded), but also fragments of the corresponding sequence of variants artificial or natural components of D11092 (or a polypeptide encoded by it). For example, the term "amino acid residues of positions 386 to 606 of a polypeptide encoded by HEV ORF2" is intended to comprise amino acid residues of positions 368 to 606 of a polypeptide encoded by D11092 and the corresponding fragments of the variants (natural or artificial ) of a polypeptide encoded by D11092. The exemplary amino acid sequence of a HEV capsid protein (a polypeptide encoded by ORF2 from D11092) is described in SEQ ID NO: 31.
[012] According to the invention, an M2 protein from the influenza virus refers to a protein encoded by the seventh type A or type B segment of the influenza virus genome or a protein encoded by the sixth segment of the genome of the influenza type C virus The exemplary amino acid sequence of an influenza virus M2 protein is described SEQ ID NO: 32.
[013] According to the invention, the expression "fragments of corresponding sequences" or "corresponding fragments" refers to fragments that are located in equal positions of the sequences when the sequences are subjected to optimal alignment, nominally, the sequences are aligned to get a higher percentage of identity.
[014] According to the invention, when used in the history of proteins / polypeptides, the term 'variant' refers to a protein, whose amino acid sequence is different from a reference protein / peptide (for example, CRM197 of the invention) by one or more (for example, 1-10, or 1-5 or 1-3) amino acids (such as conservative amino acid substitutions), or that has an identity of at least 60%, 80%, 85%, 90% , 95%, 96%, 97%, 98%, or 99% for a reference protein / polypeptide (for example, CRM197 of the invention), and which retains the essential characteristics of the reference protein / polypeptide. In the invention, the essential characteristics of CRM197 can refer to strong immunogenicity and no cytotoxicity, and the essential characteristics of a HEV capsid protein and an M2 protein of the influenza virus can refer to the antigenicity and / or immunogenicity of the same.
[015] According to the invention, the term "identity" refers to the degree of combination between two polypeptides or between two nucleic acids. When two sequences for comparison have the same base or subunit of the amino acid monomer at a given site (for example, each of the two DNA molecules has an adenine at a given site, or each of the two polypeptides has a lysine in one determined site), the two molecules are identical at the site. The percentage of identity between two sequences is a function of the number of identical sites divided by the two sequences over the total number of sites for comparison x 100. For example, if 6 out of 10 sites in the two sequences are combined, these two sequences have an identity 60%. For example, the DNA sequences: CTGACT and CAGGTT share an identity of 50% (3 of the 6 sites are combined). Generally, the comparison of two sequences is conducted in a way to produce the maximum identity. Said alignment can be conducted using a computer program such as an "Align" program (DNAstar, Inc.) which is based on the method of Needleman, et al., ("J. Mol. Biol.", 48: 443-453, 1970). The percentage of identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) that was incorporated into the “ALIGN” program ( version 2.0), using a PAM120 residue by weight table, a gap length penalty of 12 and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (“J. Mol. Biol.”, 48: 444-453 (1970)) which was incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a matrix 62 Blossum as a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
[016] As used in the invention, the term "conservative substitution" refers to amino acid substitutions that would not negatively affect or change the essential characteristics of a protein / polypeptide comprising the amino acid sequence. For example, a conservative substitution can be introduced by standard technique known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions where an amino acid residue is replaced with another amino acid residue having a similar side chain, for example, with a residue similar to the corresponding amino acid residue physically or functionally (such as, having similar size, shape , charge, chemical properties, including the ability to form and covalent bond or hydrogen bridge, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having alkaline side chains (eg, lysine, arginine and histidine), amino acids having acidic side chains (eg, lysine, arginine and histidine), amino acids having acidic side chains (eg, aspartic acid and glutamic acid) , amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine , methionine), amino acids having β-branched side chains (such as threonine, valine, isoleucine) and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, an amino acid residue is preferably substituted with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, for example, Brummell et al., "Biochem.", 32: 1180-1187 (1993); Kobayashi et al., "Protein Eng. ", 12 (10): 879-884 (1999); and Burks et al.," Proc. Natl. Acad. ", Set USA, 94: 412-417 (1997), which are incorporated herein by reference).
[017] According to the invention, the term "immunogenicity" refers to an ability to stimulate the formation of specific antibodies or sensitized lymphocytes in organisms. Not only does it refer to the property of an antigen to stimulate a specific immunocyte to activate, proliferate and differentiate the immunocyte in order to finally generate the immunological effector substance such as antibodies and sensitized lymphocytes, but it also refers to specific immune responses where the antibodies or Sensitized T lymphocytes can form an immune system in an organism after stimulating the organism with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce production and an immune response in a host depends on three factors, properties of an antigen, reactivity of a host, and the means of immunization.
[018] According to the invention, the term "immunogenic fragment" refers to said polypeptide fragment, which at least partially retains the immunogenicity of the protein from which it is derived. For example, immunogenic fragments of a HEV capsid protein refer to a fragment of a HEV capsid protein that at least partially retains immunogenicity, for example, HEV-239, E2 or E2s as described in the invention (see, Li et al ., “J. Biol. Chem.”, 2890 (5): 3400-3406 (2005); Li et al., “PLoS Pathogens”, 5 (8): e1000537 (2009); immunogenic fragments of an M2 protein from influenza virus refers to fragments of the M21 protein that at least partially retain immunogenicity, for example M2 as described in the invention (see, Fiers W. Et al., “Vaccine”, 27 (45): 6280-6283 (2009) ).
[019] According to the invention, HEV-239 (or 239 in short) refers to a polypeptide consisting of amino acid residues starting at positions 368 to 6060 of a HEV ORF2-encoded polypeptide (ie, capsid protein HEV); E2 refers to a polypeptide consisting of amino acid residues from positions 394 to 606 of a HEV ORF2 encoded polypeptide; E2s refers to a polypeptide consisting of amino acid residues from positions 455 to 606 of a HEV ORF2 encoded polypeptide.
[020] According to the invention, the term "M2e" refers to a polypeptide consisting of amino acid residues from positions 1 to 24 of an influenza virus M2 protein.
[021] In the invention, the term "polypeptide" and "protein" have the same meaning and can be used interchangeably. In addition, in the invention, amino acids are generally represented by a letter code and a three letter code well known in the art. For example, alanine can be represented by A or Ala.
[022] According to the invention, the term "expression system in E. coli", refers to an expression system consisting of E. coli (strain) and a vector, where E. coli (strain) is available on the market, including, but not limited to, GI698, ER2566, BL21 (DE3), B834 (DE3), BLR (DE3), etc.
[023] According to the invention, the term "vector" refers to a nucleic acid vehicle that can have a polynucleotide inserted into it. When the vector allows expression of the protein encoded by the polypeptide inserted into it, the vector is called an expression vector. The vector can be introduced into the host cell by transformation, transduction, or transfection, and has the elements of charged genetic material expressed in a host cell. Vectors are well known to a person skilled in the art, including, but not limited to, plasmids, phages, cosmids and the like.
[024] According to the invention, the term "chromatography" includes, but is not limited to: ion exchange chromatography (for example, cation exchange chromatography), hydrophobic interaction chromatography, absorbent chromatography (for example, hydroxyapatite chromatography) , gel filtration chromatography (gel exclusion chromatography), and affinity chromatography.
[025] According to the invention, the term "pharmaceutically acceptable vehicles and / or excipients" refers to vehicles and / or excipients that are pharmacologically and / or physiologically compatible with individuals and active ingredients, and are well known in the art (see , for example, Reminton's Pharmaceutical Sciences, Edited by Gennaro Ar, 19th edition, Pennsylvania, Mack Publishing Company, 1995), including, but not limited to, pH adjusting agents, surfactants, adjuvants, and ion extension enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers, surfactants include, but are not limited to: anionic surfactants, cationic surfactants, or nonionic surfactants (for example, Tween-80); and ionic extension enhancers include, but are not limited to, sodium chloride.
[026] According to the invention, the term "adjuvant" refers to a non-specific immuno-enhancer, which can improve the immune response to an antigen or change the type of immune response in an organism when it is released along with the antigen to the organism or is released to the organism in advance. There are a variety of adjuvants, including but not limited to, aluminum adjuvants (eg, aluminum hydroxide) Freund's adjuvant (eg, complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, and do genre. Freund's adjuvant is most commonly used in current animal experiments. The aluminum hydroxide adjuvant is most commonly used in clinical trials.
[027] According to the invention, the term "intramolecular adjuvant" refers to said adjuvant, which forms a fusion protein with a target protein (ie, an antigen) is present in the same molecule as the antigen (ie, a fusion protein comprising it in the antigen), and acts as an antigen adjuvant to improve the immunogenicity of the antigen. Nominally, an intramolecular adjuvant is an adjuvant capable of improving the immunogenicity of a target protein (antigen) fused and expressed therein, which generally refers to a polypeptide fragment. In the invention, an intramolecular adjuvant refers to a mutant non-toxic diphtheria toxin CRM197 or a fragment thereof.
[028] Techniques for formation and a fusion protein by expressing the fusion of two or more proteins well known in the art (see, for example, Sambrook J. et al., “Molecular Cloning: A Laboratory Manual” (Second Edition), Cold Spring Harbor Laboratory Press, 1989; and FM Ausubel et al., Short Protocols in Molecular Biology, 3rd edition, John Wiley & Sons, Inc., 1995). Generally, fragments of DNA encoding two or more proteins are linked together in the structure by recombinant DNA techniques, and a fusion protein is obtained by expressing the protein. Optionally, a linker can be used or not to express the fusion of two or more proteins.
[029] According to the invention, the term "linker" refers to a short peptide for binding two molecules (for example, proteins). Generally, a fusion protein, such as a target protein, 1-linker in a target protein 2, is obtained by introducing (for example, by PCR or ligase amplification) a polynucleotide encoding the short peptide between two coding DNA fragments of two target proteins to be linked, respectively, and expression of the same protein. As is well known to those skilled in the art, linkers include, but are not limited to, flexible linker peptides, such as Gly-Gly-Gly-Gly; gly-Gly-Gly-Gly-Ser; Gly-Gly-Ser-Ser and (Gly-Gly-Gly-Gly-Ser) 3.
[030] According to the invention, the term "an effective amount" refers to an amount that is sufficient to achieve or at least partially achieve the expected effect. For example, an effective amount to prevent a disease (such as a HEV or influenza virus infection) refers to an effective amount to prevent, suppress, or delay the occurrence of a disease (such as HEV or an influenza virus infection). An effective amount to treat a disease refers to an effective amount to cure or at least partially block a disease and its complication in a patient with the disease. The determination of the said effective quantity is within the capacity of a technician in the matter. For example, an effective amount for a therapeutic use depends on the severity of a disease to be treated, general condition of the immune system in a patient, general conditions of a patient, such as age, weight and sex, means of drug administration, therapies additional used simultaneously, and the gender.
[031] The invention is at least partially based on the surprising discovery of the inventors: after the expression of the CRM197 fusion or a fragment thereof with a target protein (for example, a HEV capsid protein, an M2 protein from the influenza virus or an immunogenic fragment thereof), CRM197 or a fragment thereof significantly improves the immunogenicity of the target protein. Nominally, CRM197 or a fragment thereof can be used as an intramolecular adjuvant to improve the immunogenicity of a target protein by expressing the fusion with the target protein.
[032] Therefore, in one aspect, the invention relates to a fusion protein comprising CRM197 or a fragment thereof in a target protein, said CRM197 or a fragment thereof improving the immunogenicity of the target protein.
[033] In a preferred embodiment, the CRM197 fragment comprises, for example, the catalytic domain (aa 1-190, also called fragment A in the present application), transmembrane T domain (aa 201-384), and / or domain CRM197 receptor-binding R (aa 386-535). For example, the CRM197 fragment can comprise fragment A, or fragment a and the transmembrane domain T.
[034] In another preferred embodiment, the CRM197 fragment comprises aa 1-190 of CRM197, for example, comprises aa 1-389 of CRM197. In another preferred embodiment, the CRM197 fragment consists of aa 1-190 or aa 1-389 of CRM197. In the present patent application, the exemplary amino acid sequence of CRM197 is represented by SEQ ID NO: 2, and the corresponding nucleotide sequence is represented in SEQ ID NO: 1.
[035] In a preferred embodiment, the target protein can be a HEV capsid protein, an M2 protein from the influenza virus, or an immunogenic fragment thereof. In another preferred embodiment, the immunogenic fragment of a HEV capsid protein may comprise or be, for example, HEV-239 (aa 368-606 of the HEV capsid protein), E2 (aa 394-606 of the HEV capsid protein) or E2s (aa 455-606 of HEV capsid protein), and the genus. In another preferred embodiment, the immunogenic fragment of an M2 protein can comprise or be, for example, M2e (aa 124 of the M2 protein).
[036] In a preferred embodiment, the fusion protein of the invention CRM197 or a fragment thereof can be linked to an N and / or C terminal of the target protein, optionally via a linker. The linker for the binding of two peptide fragments is well known in the art, including but not limited to flexible binding peptides, such as, Gly-Gly-Gly, Gly-Gly-Gly-Gly-Ser, Gly-Gly -Ser-Ser e (Gly-Gly-Gly-Gly-Ser) 3, etc. The said binders are well known in the art, and the selection of the same is within the skill of a person skilled in the art.
[037] In a preferred embodiment, the fusion protein of the invention may comprise CRM197 or a fragment thereof, and a HEV capsid protein or an immunogenic fragment thereof, which are linked together, optionally via a linker. For example, the fusion protein of the invention can be a protein having an amino acid sequence represented in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18.
[038] In a preferred embodiment, the fusion protein of the invention can comprise CRM197 or a fragment thereof, and an M2 protein from the influenza virus or an immunogenic fragment thereof, which are optionally linked together via a linker. For example, the fusion protein of the invention can be a protein having an amino acid sequence represented in SEQ ID NOs: 34, 36, 38, 40, 42 or 44.
[039] In the CRM197 fusion protein of the invention or a fragment thereof, it surprisingly increases the immunogenicity of a target protein (such as HEV capsid protein, an M2 protein from the influenza virus or an immunogenic fragment thereof) fused to it. (optionally via a linker) significantly, and thus, can be used as an intramolecular adjuvant.
[040] In another aspect, the invention provides a polynucleotide encoding the fusion protein as defined above, and also provides a construct comprising the polynucleotide.
[041] In another aspect, the invention provides a vector comprising: a polynucleotide encoding the fusion protein as defined above or a construct comprising the polynucleotide. The invention vector can be a cloning vector, or an expression vector.
[042] In a preferred embodiment, the vector of the invention can be, for example, a plasmid, cosmids, phage, and the like.
[043] In another aspect, the invention provides a host cell or organism comprising the polynucleotide, the construct, or the vector of the invention. Said host cell includes, but is not limited to, prokaryotic cell, such as E. coli cell, and eukaryotic cell such as yeast cell, insect cell, plant cell and animal cells (such as mammalian cell, eg mouse cell, human cell and gender). The cell of the invention can be a cell line, such as a 293T cell. In one embodiment, the organism is a plant or animal.
[044] In another aspect, the invention also relates to a pharmaceutical composition or vaccine comprising the fusion protein of the invention and, optionally, a pharmaceutically acceptable carrier and / or excipient. Depending on the target protein used in the fusion protein, the pharmaceutical composition or vaccine of the invention can be useful for the prevention and / or treatment of various diseases (i.e., diseases that can be prevented or treated by the target protein). For example, when the target protein used is a HEV capsid protein or an immunogenic fragment thereof, the pharmaceutical composition of the invention can be used to prevent and / or treat HEV infection and diseases associated with HEV infection such as Hepatitis E ; when the target protein used is an influenza virus M2 protein or a target immunogenic fragment, the pharmaceutical composition of the invention can be used to prevent and / or treat influenza virus infection and diseases associated with influenza virus infection such as influenza .
[045] In another aspect, the invention also relates to the use of the fusion protein of the invention in the manufacture of a pharmaceutical composition for the prevention and / or treatment of diseases that can be prevented or treated by the target protein. Depending on the target protein used in the fusion protein, the pharmaceutical composition of the invention can be used to prevent and / or treat various diseases. For example, when the target protein used is a HEV capsid protein or an immunogenic fragment thereof, the pharmaceutical composition of the invention can be used to prevent and / or treat HEV infection and diseases associated with HEV infection such as Hepatitis E; when the target protein used is an influenza virus M2 protein or an immunogenic fragment thereof, the pharmaceutical composition of the invention can be used to prevent and / or treat influenza virus infection and diseases associated with influenza virus infection such as influenza .
[046] In another aspect, the invention also relates to a method for preventing HEV infection and / or diseases associated with HEV infection such as Hepatitis E, comprising administering an effective amount of the invention fusion protein or pharmaceutical composition comprising the fusion protein, the fusion protein comprising CRM197 or a fragment thereof and a HEV capsid protein or an immunogenic fragment thereof, which are optionally linked together via a linker.
[047] In another aspect, the invention also relates to a method for preventing and / or treating influenza virus infection and diseases associated with influenza virus infection such as influenza, comprising administering an effective amount of the influenza protein. fusion of the invention or the pharmaceutical composition comprising the fusion protein, the fusion protein comprising CRM197 or a fragment thereof and an M2 protein from the influenza virus or an immunogenic fragment thereof, which are optionally linked together via a linker .
[048] In another aspect, the invention provides a method for improving the immunogenicity of a target protein, comprising obtaining a fusion protein containing CRM197 or a fragment thereof as defined above and the target protein, in order to improve immunogenicity of the target protein.
[049] In a preferred embodiment, the fusion protein can be obtained by expressing the fusion of CRM197 or a fragment thereof with the target protein, optionally using a linker. In a preferred embodiment, the target protein is the HEV capsid protein, the M2 protein from the influenza virus or an immunogenic fragment thereof as described above.
[050] Therefore, in one embodiment, the invention provides a method for improving the immunogenicity of a HEV capsid protein or an immunogenic fragment thereof, comprising obtaining a fusion protein, comprising CRM197 or a fragment thereof and a protein HEV capsid or an immunogenic fragment thereof, in order to improve the immunogenicity of the HEV capsid protein or an immunogenic fragment thereof. In a preferred embodiment, the fusion protein can be obtained by expressing the fusion of CRM197 or a fragment thereof with a HEV capsid protein or an immunogenic fragment thereof, optionally using a linker.
[051] In another embodiment, the invention provides a method for improving the immunogenicity of an M2 protein from the influenza virus or an immunogenic fragment thereof, comprising obtaining a fusion protein containing CRM197 or a fragment thereof and an M2 protein influenza virus or an immunogenic fragment thereof, in order to improve the immunogenicity of the influenza virus M2 protein or an immunogenic fragment thereof. In a preferred embodiment, the fusion protein can be obtained by expressing the fusion of CRM197 or a fragment thereof with an M2 protein from the influenza virus or an immunogenic fragment thereof, optionally using a linker.
[052] In another aspect, the invention relates to the use of CRM197 or a fragment thereof in improving the immunogenicity of a target protein, characterized by obtaining a fusion protein containing CRM197 or a fragment thereof and the target protein.
[053] In a preferred embodiment, the fusion protein can be obtained by the expression of fusion of CRM197 or a fragment thereof with the target protein, optionally using a linker. In a preferred embodiment, the target protein is a HEV capsid protein, an M2 protein from influenza virus, or an immunogenic fragment thereof.
[054] Therefore, in one embodiment, the invention relates to the use of a CRM197 or a fragment thereof in improving the immunogenicity of a HEV capsid protein or an immunogenic fragment thereof, characterized by obtaining a fusion protein comprising CRM197 or a fragment thereof and the HEV capsid protein or an immunogenic fragment thereof. In a preferred embodiment, the fusion protein can be obtained by expressing the fusion of CRM197 or a fragment thereof with the HEV capsid protein or an immunogenic fragment thereof, optionally using a linker.
[055] In another embodiment, the invention relates to the use of CRM197 or a fragment thereof in improving the immunogenicity of an M2 protein from the influenza virus or an immunogenic fragment thereof, characterized by the fact that it obtains a fusion protein comprising CRM197 or a fragment thereof and the M2 protein of the influenza virus or an immunogenic fragment thereof. In a preferred embodiment, the fusion protein can be obtained by expressing the fusion of CRM197 or a fragment thereof with the M2 protein from the influenza virus or an immunogenic fragment thereof, optionally using a linker. Beneficial effects of the invention
[056] The invention demonstrates in a first moment that CRM197 and its fragments can be used as intramolecular adjuvants to improve the immunogenicity of a target protein. Therefore, the invention provides a new use of CRM197 and fragments thereof, and provides a new method for improving the immunogenicity of a target protein.
[057] In addition, since the fusion protein of the invention has a strong immunogenicity when compared to a target protein alone, the invention provides a new option for the manufacture of a medicine or vaccine and can achieve a more effective treatment and prevention of corresponding diseases.
[058] For example, the fusion protein of the invention comprising CRM197 (or a fragment thereof) and a HEV capsid protein (or an immunogenic fragment thereof) exhibits strong immunogenicity when compared to a HEV capsid protein (or a immunogenic fragment thereof) and, therefore, the fusion protein can be useful for the manufacture of a pharmaceutical composition and, more effectively prevent and treat HEV infection and diseases associated with HEV infection, such as Hepatitis E.
[059] For example, the fusion protein of the invention comprising CRM197 (or a fragment of it) and an M2 protein from the influenza virus (or an immunogenic fragment thereof) exhibits strong immunogenicity when compared to an M2 protein from the influenza virus ( or an immunogenic fragment thereof) and, therefore, the fusion protein can be useful for the manufacture of a pharmaceutical composition and, more effectively, prevent and treat influenza virus infection and diseases associated with influenza virus infection, such as , influenza. For example, when the M2e protein is fused to the CRM197 N-terminal (or a fragment thereof), the fusion protein thus formed can form a tetramer or other polymer configuration, and has good reactivity with a protective O19 monoclonal antibody. (see, Fu et al., “Virology”, 2009, 385: 218-226) in vitro (see, figure 12B), and has good immunogenicity, in vivo (see, figure 14). Therefore, the fusion protein thus formed is useful for the general development of influenza vaccines. Description of the sequence information
[060] The sequence information as involved in the invention is provided below in the table:



[061] Embodiments of the invention are further described in detail with reference to the drawings and examples. However, a person skilled in the art would understand that the following drawings and examples are intended to illustrate the invention only, in addition to defining the scope of the invention. According to the detailed description of the following drawings and preferred embodiments, various purposes and advantages of the invention would be obvious to one skilled in the art. Description of the drawings
[062] Figure 1 illustrates the clone design of the fusion proteins constructed in Example 2, with the linker used (linker, also referred to as L for short in the present application) being a flexible fragment consisting of 15 amino acid residues, whose sequence is GGGGSGGGGSGGG; the CRM197 used comprises 535 amino acids, the sequence of which is represented in SEQ ID NO: 2; 389 refers to a polypeptide comprising amino acid residues from positions 1 through 389 (aa 1-389) of CRM197; A refers to a polypeptide comprising amino acid residues from positions 1 to 190 (aa 1-190) of CRM197; E2 refers to a polypeptide comprising amino acid residues from positions 394 to 606 (aa 394-606) of a HEV capsid protein; E2s refers to a polypeptide comprising amino acid residues from positions 455 to 606 (aa 455-606) of a HEV capsid protein;
[063] Figure 2 illustrates the SDS-PAGE analytical results of the expression, purification and renaturation of the fusion proteins constructed in Example 2, the sample used in Figure 2A being the precipitate (that is, inclusion body) obtained by centrifugation. of the broken bacteria after ultrasonication, the sample used in figure 2B was a supernatant dissolved in 4M urea, the sample used in figure 2C was a supernatant dissolved in 8M urea, and the sample used in figure 2D was a renatured protein inside PBS. Column M: molecular weight marker; Column 1; CRM197-L-E2; Column 2: CRM197-L-E2s; Column 3: 389-L-E2; Column 4: 389-L-E2s; Column 5: 389-E2s; Column 6: A-L-E2; Column 7: A-L-E2s; Column 8: A-E2s. The results illustrated that all the fusion proteins constructed could be expressed in inclusion bodies, and A-L-E2 and A-L-E2s were dissolved in 4M and 8M urea, while other fusion proteins were only dissolved in 8M urea. In addition, the results also showed that after dialysis and renaturation, fusion proteins of a purity of about 80% were obtained;
[064] Figure 3 illustrates the SDS-PAGE result of the AL-E2 fusion protein purified by chromatography, with column 1 referring to AL-E2 which is renatured to PBS after purification by chromatography, column 2 refers to to an AL-E2 sample from column 1 boiled in boiling water for 10 minutes. The results showed that after two chromatography steps, A-L-E2 could achieve a purity of about 90%;
[065] Figure 4 illustrates the results of a Western blotting using the fusion proteins constructed in Example 2 and the neutralizing monoclonal antibody HEV 8C11. Column M: protein molecular weight marker; column 1: HEV-239 control protein; Column 2: Control Protein E2, Column 3: CRM197-L-E2; Column 4: CRM197-L-E2s; Column 5: 389-L-E2; Column 6: 389-L-E2s; Column 7: 389-E2s; Column 8: A-L-E2; Column 9: A-L-E2s; Column 10: A-E2s. The results showed that all the fusion proteins tested had significant reactivity with the specific neutralizing monoclonal antibody HEV 8C11;
[066] Figure 5 illustrates the direct results of the ELISA, using the fusion proteins constructed in Example 2, and the specific monoclonal antibody HEV. The abscissa refers to the specific monoclonal antibody HEV or polyclonal antiserum CRM197 for ELISA, and the ordinate refers to the OD value determined by the ELISA at the same dilution as the antibody. Figure 5A illustrates the ELISA result of the fusion protein comprising E2, and Figure 5B illustrates the ELISA result of the fusion proteins comprising E2s. The results showed that the reactivity of the E2s protein with the specific monoclonal antibody HEV was significantly improved, after the fusion of the E2s protein with CRM197 or a fragment thereof, with the reactivity of AL-E2s and A-E2S being more significantly improved , the reactivity of the E2 protein with the specific monoclonal antibody HEV was retained or improved, after the E2 fusion protein with CRM197 or a fragment thereof;
[067] Figure 6 shows indirect ELISA results, using proteins A-L-E2, HEV-239 or E2 and specific monoclonal antibody HEV, with the cut-off value being defined as three times the mean negative value. The results showed that the reactivity of A-L-E2 with the specific monoclonal antibody HEV is comparable to that of HEV-239 and E2;
[068] Figure 7 illustrates the analytical result of the sedimentation rate (SV) of the A-L-E2 fusion protein. The result showed that the A-L-E2 fusion protein was mainly present in a dimer form, and tetramer which is present in a smaller amount;
[069] Figure 8 illustrates the comparison of immunogenicity between the fusion proteins constructed in Example 2 and HEV-239. Primary immunization was performed at week 0, and induction immunization was performed at week 2 and week 4, with the dose for both primary immunization and induction immunization being 5 μ g or 0.5 μ g . Figure 8 A shows the result of the comparison of the immune serum antibody titration in 5μg dose groups, and Figure 8B illustrates the result of the comparison of the immune serum antibody titration in 0.5μg dose groups. The results showed that the serum conversion against HEV occurred in the mouse serum at week 4 in groups of doses of 5μg and 0.5μg, and the antibody titration reached the highest value at week 5 or 6. In particular, in the group at a dose of 5μ g, the highest antibody titre was obtained when AL-E2 was used, which reached 106 in week 6; and the antibody titers induced by the fusion proteins were greater than or comparable to that of the HEV-239 protein. In the 0.5μg dose groups, the antibody titer of the fusion proteins was significantly higher than that of HEV-239, and the antibody titre induced by the AL-E2 protein at week 5 reached 106. In addition, the Serum conversion did not occur in the immune serum when using E2 and E2s in the 5μg and 0.5μg dose groups. as seen from the results above, the immunogenicity of the fusion proteins constructed in Example 2 were significantly higher than the antigen protein (E2 and E2s) alone, indicating that the CRM197 of the invention or a fragment thereof significantly improved the immunogenicity of the antigen protein fused to it, and could be used as an intramolecular adjuvant;
[070] Figure 9 illustrates the design of the fusion protein clone constructed in Example 6, with the linker used (linker, also referred to as L for short in the present application) being a flexible fragment consisting of 10 amino acid residues, whose sequence is GGGGSGGGGS; the CRM197 used comprising 535 amino acids, the sequence of which is represented in SEQ ID NO: 2; 389 refers to a polypeptide comprising amino acid residues from positions 1 to 389 (aa 1-389) of CRM197; A refers to a polypeptide comprising amino acid residues from positions 1 to 190 (aa 1-190) of CRM197; M2 refers to an influenza virus M2 protein, the sequence of which is represented in SEQ ID NO: 32; M2e refers to a polypeptide comprising amino acid residues from positions 1 to 24 (aa 1-24) of the influenza virus M2 protein;
[071] Figure 10 illustrates the SDS-PAGE analytical results of the expression, purification and renaturation of the fusion proteins constructed in Example 6, with column M: the protein molecular weight marker;
[072] Figure 10A used the samples that were precipitated (ie, inclusion body) and the supernatant obtained by centrifuging the broken bacterium after ultrasonication: Column 1: inclusion body obtained from the bacteria transformed with CRM197-L-M2e ; Column 2: supernatant obtained from the bacteria transformed with CRM197-L-M2e; Column 3: inclusion body obtained from bacteria transformed with 389-L-M2e; Column 4: supernatant obtained from bacteria transformed with 389-L-M2e; Column 5: inclusion body obtained from the bacteria transformed with A-L-M2e; Column 6: supernatant obtained from the bacteria transformed with A-L-M2e;
[073] Figure 10B used the samples that were precipitated (that is, inclusion bodies), and the supernatant obtained by centrifuging the broken bacteria after ultrasonication; Column 1: inclusion body obtained from bacteria transformed with M2e-L-A; Column 2: supernatant obtained from the bacteria transformed with M2e-L-A; Column 3: inclusion body obtained from the bacteria transformed with M2e-L-389; Column 4: supernatant obtained from the bacteria transformed with M2e-L-389; Column 5: inclusion body obtained from the bacteria transformed with M2e-L-CRM197; Column 6: supernatant obtained from the bacteria transformed with M2e-L-CRM197;
[074] Figure 10C used the samples that were obtained from the isolated and renatured fusion proteins in PBS, with no β-mercaptoethanol being used during the SDS-PAGE analysis, and the protein samples were treated by ebolition (for 10 minutes ) or not: Column 1: AL-M2e protein, not treated by boiling; Column 2: protein A-L-M2e, treated by boiling; Column 3: 389-L-M2e protein, untreated by boiling; Column 4: 389-L-M2e protein, treated by boiling; Column 5: CRM197-L-M2e protein, untreated by boiling; Column 6: CRM197-L-M2e protein, treated by boiling;
[075] Figure 10D used the samples that were obtained from the isolated and renatured fusion proteins in PBS, with β-mercaptoethanol being used during the SDS-PAGE analysis, and the protein samples boiled (for 10 minutes) or not: Column 1: AL-M2e protein, not treated by boiling; Column 2: protein A-L-M2e, treated by boiling; Column 3: 389-L-M2e protein, untreated by boiling; Column 4: 389-L-M2e protein, treated by boiling; Column 5: CRM197-L-M2e protein, untreated by boiling; Column 6: CRM197-L-M2e protein, treated by boiling;
[076] Figure 10E used the samples that were obtained from the isolated and renatured fusion proteins in PBS, with no β-mercaptoethanol being used during the SDS-PAGE analysis, and the samples of the protein treated by boiling (for 10 minutes ) or not: Column 1: M2e-LA protein, not treated by boiling; Column 2: M2e-L-A protein, treated by boiling; Column 3: M2e-L-389 protein, untreated by boiling; Column 4: M2e-L-389 protein, treated by boiling; Column 5: M2e-L-CRM197 protein, untreated by boiling; Column 6: M2e-L-CRM197 protein, treated by boiling;
[077] Figure 10F used the samples that were obtained from the isolated and renatured fusion proteins in PBS, with β-mercaptoethanol being used during SDS-PAGE analysis, and the protein samples boiled (for 10 minutes) or not: Column 1: M2e-LA protein, not treated by boiling; Column 2: M2e-L-A protein, treated by boiling; Column 3: M2e-L-389 protein, untreated by boiling; Column 4: M2e-L-389 protein, treated by boiling; Column 5: M2e-L-CRM197 protein, untreated by boiling; Column 6: M2e-L-CRM197 protein, treated by boiling;
[078] The results shown in figures 10 A - 10F indicated that all constructed fusion proteins could be expressed in inclusion bodies and, after purification and renaturation, fusion proteins with a purity of about 80% could be obtained ;
[079] Figure 11 illustrates the results of Western blotting using the fusion proteins constructed in Example 6 and the anti-M2e monoclonal antibody 5D1 and the monoclonal antibody 1E6 CRM197. The samples represented by columns 1 - 6 in figures 11A, 11B, 11C and 11D correspond to the samples represented by columns 1-6 in figures 10C, 10D, 10E and 10F, respectively; the specific monoclonal antibody anti-M2e 5D1 was used. The samples represented by columns 1-6 in figures 11E, 11F, 11G and 11H correspond to the samples represented by columns 1-6 in figures 10C, 10D, 10E and 10F, respectively, with the specific monoclonal antibody CRM197 1E6 being used. The results showed that all the fusion proteins tested had significant reactivity with the specific monoclonal antibody anti-M2e 5D1 and the specific monoclonal antibody CRM197 1E6;
[080] Figure 12 illustrates the indirect results of the ELISA, using the fusion proteins constructed in Example 6 and several specific anti-M2e monoclonal antibodies. abscissa refers to specific anti-M2e monoclonal antibodies and specific anti-CRM197 monoclonal antibodies to ELISA, and ordinate refers to the OD value determined by the ELISA at the same dilution as the antibody. Figure 12A illustrates the ELISA result of the fusion protein in which M2e was fused to the CRM197 C-terminus or a fragment thereof, and Figure 12B illustrates the ELISA result of the fusion protein in which M2e was fused to N - terminal of CRM197 or a fragment thereof. The results showed that the fusion protein comprising the M2e protein and the CRM197 or the fragment thereof retained or improved reactivity with several specific anti-M2e monoclonal antibodies, when compared to the M2e protein alone;
[081] Figure 13 shows the analytical results of the sedimentation rate (SV) of the fusion proteins constructed in example 6, with Figure 13A: CRM197-L-M2e; Fig. 13B: 389-L-M2e; Fig. 13C: A-L-M2e; Fig. 13D: M2e-L-CRM197; Fig. 13E: M2e-L-389; Fig. 13F: M2e-L-A. The results showed that the fusion protein A-L-M2e and M2e-L-A were mainly present in a monomer and tetramer form; and 389-L-M2e was mainly present in a form of dimer and polymer; M2e-L-389 was mainly present in a monomer and polymer form; CRM197-L-M2e was mainly present in a dimer and polymer form; and M2e-L-CRM197 was mainly present in the form of monomer and polymer; and
[082] Figure 14 illustrates the comparison of immunogenicity between the fusion proteins constructed in Example 6 and GST-M2e. Primary immunization was performed at week 0, and induction immunization was performed at week 2 and 4, with the dose for both primary immunization and induction immunization being 5 μ g or 0.5 μ g. Figure 14 A illustrates the result of comparing the antibody titer in the immune serum in 0.5 μ g dose groups. As seen in the results above, the immunogenicity of the fusion proteins constructed in Example 6 was significantly greater than the antigen protein (GST-M2e) alone, indicating that the CRM197 of the invention or a fragment thereof (no matter located at the N-terminus) or C-terminus of the fusion protein) significantly improved the immunogenicity of the antigen protein fused to it, and could be used as the intramolecular adjuvant. Specific forms for carrying out the invention
[083] The present invention is illustrated with reference to the following examples (which are used only for the purpose of illustrating the present invention and are not intended to limit the scope of protection of the present invention).
[084] Unless otherwise indicated, the molecular biological experimental methods and immunological analysis used in the present invention are performed substantially in accordance with the methods described in Sambrook J. et al., “Molecular Cloning: A Laboratory Manual (second edition) ), Cold Spring Harbor Laboratory Press ”, 1989, and FM Ausubel et al., Short Protocols in Molecular Biology”, 3rd edition, John Wiley & Sons, Inc., 1995; Restriction endonucleases are used under the conditions recommended by the product manufacturer. The reagents used in the present invention, whose manufacturers are clearly indicated, are products conventional in the art or commercially available. Those skilled in the art understand that the examples are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Example 1: CRM197 gene clone
[085] Genomic DNA extracted from the strain of Diphteria bacillus C7 (β197) obtained from the ATCC (No. 53281) was used as a standard for the PCR reaction, with the front primer being CRM197F (SEQ ID NO: 19) , and the reverse primer was CRM197R (SEQ ID NO: 20). The PCR reaction was performed in a PCR device (Biometra T3) under the following conditions, to prepare the full length gene encoding CRM197. 94 ° C denaturing 10 min 1 cycle 94 ° C denaturing 1.5 minutes 20 cycles 58 ° C annealing 1.5 minutes 72 ° C prolongation 1.5 minutes 72 ° C prolongation - 10 minutes 1 cycle

[086] After PCR amplification, a product of about 1.6 kb in length was obtained. After sequencing, the nucleotide sequence (SEQ ID NO: 1) of the amplification product (i.e., the full length gene for CRM197) was obtained, and the amino acid sequence encoded in this way was represented in SEQ ID NO: 2 . Example 2:
[087] Design and clone of the fusion proteins comprising CRM197 or a fragment thereof and a fragment of the HEV capsid protein.
[088] In the example, the vectors expressing the fusion protein were constructed exemplarily. The clone design of several examples of constructed fusion protein is shown in Figure 1, with each fusion protein comprising CRM197 or a fragment thereof and a HEV capsid protein fragment optionally, using a linker.
[089] A fusion protein clone comprising a linker:
[090] The amplification product (i.e., the full length gene from CRM197) obtained in Example 1 was used as a template. The previous primer was CRM197F (SEQ ID NO: 19), at the 5 'end from which the CAT ATG restriction endonuclease NdeI site was introduced, with ATG being the start codon in the E. coli system. The reverse primers were CRM197-linker R (SEQ ID NO; 21), 389-linker R (SEQ ID NO; 22) and A-linker R (SEQ ID NO: 23), respectively, at the 5 'end of which the site BamHI from the GGA TCC restriction endonuclease was introduced. The PCr reaction was performed in a PCR thermocycler (Biometra T3) under the following conditions. The sequences of the primers used are shown in table 1. 94 ° C denaturing 10 min 1 cycle 94 ° C denaturing 1.5 minutes 20 cycles 58 ° C annealing 1.5 minutes 72 ° C prolongation - 1.5 minutes 72 ° C prolongation - 10 minutes 1 cycle

[091] The amplification products were DNA fragments of about 1600bp, 1200 bp and 600 bp in length, respectively.
[092] In addition, pTO-T7-E2 (Li, et al., JBC.2005, 28 (5): 3400-3406) was used as a standard. The previous primers were E2F (SEQ ID NO: 24) and E2sF (SEQ ID NO: 25), respectively at the 5 'terminal from which the BamHI site of the GGA TCC restriction endonuclease was introduced. The reverse primer was Drp59R (SEQ ID NO: 26), at the 5 'terminal from which the GARI TTC restriction endonuclease EcoRI site was introduced. The PCR reaction was performed in a PCR thermocycler (Biometra T3) under the following conditions. 94 ° C denaturation 10 min 1 cycle 94 ° C denaturation 50 seconds 20 cycles 58 ° C annealing 58 seconds 72 ° C extension 50 seconds 72 ° C extension - 10 minutes 1 cycle

[093] The amplification products were DNA fragments about 600 bp and 450 bp in length, respectively.
[094] The amplification products as obtained above were linked in a commercially available pMD 18-T vector (produced by TAKARA Co.), respectively, and designated as pMD 18-T-CRM197-L, pMD 18-T-389- L and pMD 18-TAL as well as pMD 18-T-E2 and pMD 18-T-E2s. As identified by the cleavage enzymes NdeI / BamHI and BamHI / EcoRI, positive clones pMD 18-T-CRM197-L, pMD 18-T-389-L, pMD 18-TAL, pMD 18-T-E2 and pMD, respectively 18-T-E2 were obtained.
[095] As confirmed by the M13 (+) primer, the correct nucleotide sequences of interest were inserted into pMD 18-T-CRM197, pMD 18-T-389-L, pMD 18-TAL, pMD 18-T-E2 and pMD 18-T-E2s, respectively.
[096] Plasmids pMD 18-T-CRM197-L, pMD 18-T-389-L and pMD 18-T-A-L were digested with the enzyme NdeI / BamHI. The fragments obtained by the cleavage enzyme were ligated into a prokaryotic expression vector pTO-T7 digested by the enzyme NdeI / BamHI (Luo Wenxin et al., “Chinese Journal of Biotechnology”, 2000, 16: 53-57), and were transformed E. coli ER2566 (purchased from Invitrogen Co.); after extraction of the plasmids, as identified by the NdeI / BamHI cleavage enzymes, the positive plasmids pTO-T7-CRM197-L, pTO-T7- 389-L and pTO-T7-AL, within which CRM197-L, 389-L and AL were inserted, respectively, were obtained.
[097] pTO-T7-CRM197-L, pTO-T7-389-L, pTO-T7-A-L, pMD 18-T-E2 and pMD 18-T-E2s were digested by the enzyme BamHI / EcoRI. Each obtained from fragments E2 and E2s was ligated into vectors pTO-T7-CRM197-L, pTO-T7-389-L and pTO-T7-A-L digested by the enzyme BamHI / EcoRI, respectively. As identified by the NdeI / EcoRI cleavage enzyme, the positive expression vectors pTO-T7-CRM197-L-E2, pTO-T7- CRM197-L-E2s, pTO-T7-389-L-E2, pTO-T7-389 -L-E2s, pTO-T7-A- L-E2 and pTO-T7-AL-E2s, within which CRM197-L-E2 (SEQ ID NO: 3, 4), CRM197-L-E2s (SEQ ID NO : 5, 6), 389-L-E2 (SEQ ID NO: 7, 8), 389-L-E2s (SEQ ID NO: 9, 10), AL-E2 (SEQ ID NO: 11, 12) or AL -E2s (SEQ ID NO: 13, 14) was inserted, respectively, were obtained. Clone of the 389-E2s fusion proteins without a ligand:
[098] The vectors expressing 389-E2s and A-E2s were constructed by three PCR reactions. For the first reaction and PCR, the CRM197 full extension gene was used as a template. The first previous one was CRM197F, at the 5 'terminal from which the CAT ATG restriction endonuclease NdeI site was introduced, with ATG being the initial codon in the E. coli system. The reverse primer forms 389-E2s R (SEQ ID NO: 27) and A-E2s R (SEQ ID NO: 28) respectively. Amplification was performed to obtain the N-terminal fragments of the fusion proteins.
[099] For the second PCR reaction, the CRM197 full extension gene was used as a template. The previous primers were 389-E2s (SEQ IDN O; 29) and A-E2s F (SEQ ID NO: 30), respectively. The reverse primer was DrP59 R, at the 5 'end from which the GARI TTC restriction endonuclease EcoRI site was introduced. Amplification was performed to obtain the C-terminal fragments of the fusion protein. The first and second PCR reactions were performed in a PCR thermocycler (Biometra T3) under the following conditions: 94 ° C denaturation 10 min 1 cycle 94 ° C denaturation 50 sec 20 cycles 58 ° C annealing 50 sec 72 ° C extension 50 sec 72 ° C extension 10 min 1 cycle
[100] For the third PCR reaction, the amplification products of the first and second reaction and PCR were used as a template (for example, the two fragments obtained by using 389-E2sF and 389-E2sR as primers were used as a template for amplification of 389-E2s), and CRM197F and DrP59R were used as primers. Amplification was performed in a PCR thermocycler (Biometra T3) under the following conditions: 94 ° C denaturation 10 min 1 cycle 94 ° C denaturation 50 sec 20 cycles 58 ° C annealing 50 sec 72 ° C extension 50 sec 72 ° C prolongation 10 min 1 cycle
[101] The amplification products were fragments of DNA about 1600 bp and 1000 bp in length, respectively. The amplification products obtained above were linked within the commercially available 18-T vector (produced by TAKARA Co.), respectively. As identified by the NdeI / EcoRI cleavage enzyme, positive clones pMD 18-T-389-E2s and pMD 18-T-A-E2s were obtained.
[102] As confirmed by the M13 (+) primer, the correct nucleotide sequences of SEQ IDN O: 15 and SEQ ID NO: 17 (which encode the amino acid sequence of SEQ ID NO; 16 and SEQ ID NO: 18, respectively ) were inserted into pMD 18-T-389-E2s and pMD 18-TA-E2s, respectively.
[103] Plasmids pMD 18-T-389-E2s and pMD 18-T-A-E2s were digested by the enzyme NdeI / EcoRI. The fragments obtained by the cleavage enzyme were then ligated into the prokaryotic expression vector pTO-T7 digested by the enzyme NdeI / EcoRI (Luo Wenxin et al., “Chinese Journal of Biotechnology”, 2000, 16: 53-57). As identified by the NdeI / EcoRI cleavage enzyme, the positive plasmids pTO-T7-389-E2s and pTO-T7-A-E2s, into which 389-E2s and A-E2s were inserted, respectively, were obtained.
[104] The sequences of the primers used in the Example are shown in Table 1. Table 1: Primer sequences


[105] 1μL of plasmids pTO-T7-CRM197-L-E2, pTO-T7-CRM197- L-E2s, pTO-T7-389-L-E2, pTO-T7-389-L-E2s, pTO-T7- 389-E2s, pTO-T7-AL-E2, pTO-T7-AL-E2s and pTO-T7-A-E2s (0.15mg / ml) were separately used to transform 40 μL of competent E. coli ER2566 (purchased at Invitrogen) prepared by the calcium chloride method, and then the bacteria were plated on solid LB medium (the components of the LB medium: 10 g / L of peptone, 5 g / L of yeast powder, and 10 g / L of NaCl , the same below) containing kanamycin (at a final concentration of 100 mg / ml, the same below). The plates were statically incubated at 37 ° C for about 10-12 hours until the individual colonies were clearly seen. The individual colonies of the plates were transferred to a tube containing 4 ml of liquid LB medium containing kanamycin.
[106] Cultures were incubated in a shaking incubator at 180 rpm for 10 hours at 37 ° C, and then 1 ml of bacterial solutions were taken and stored at - 70 ° C. Example 3: Expression and purification of the fusion proteins constructed in Example 2. Expression of the fusion proteins and purification of the inclusion bodies:
[107] 5μl of the bacterial solution, taken from an ultra-low freezer temperature at -70 ° C, was seeded in 5 ml of liquid LB medium containing kanamycin, and then it was grown at 37 ° C, 180 rpm under agitation until OD600 reached about 0.5. The resulting solution was transferred to 500 ml of LB medium containing kanamycin, and then it was grown at 37 ° C, 180 rpm with stirring for 4-5 hours. When OD600 reached about 1.5, IPTG was added to a final concentration of 0.4 mM, and the bacteria were induced under stirring at 37 ° C for 4 hours.
[108] After induction, centrifugation was performed at 8000g for 5 minutes to collect the bacteria, and then the bacteria were resuspended in a lysis solution in a ratio of 1g of bacteria to 10 ml of lysis solution (20 mM Tris buffer, pH 7.2; 300 mM NaCl), in an ice bath. The bacteria were treated with a sonicator (Sonics VCX750 Type sonicator) (Conditions: operating time 15 minutes, pulse 2 seconds, intermissions of 4 seconds, output energy 55%). The bacterial lysate was centrifuged at 12000 rpm, 4 ° C for 5 minutes (the same below), the supernatant was discarded and the precipitate (ie, the inclusion bodies) was maintained; 2% Triton-100 of the same volume was used for washing, the resulting mixture was vibrated for 30 minutes, centrifuged, and the supernatant was discarded. The precipitate was resuspended in Buffer I (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM EDTA), under vibration for 30 minutes, centrifuged, and the supernatant was discarded. The precipitate was then resuspended in 2M urea, under vibration at 37 ° C for 30 minutes, centrifuged, the supernatant and the precipitate were obtained. The supernatant was maintained; and the precipitate was resuspended in 4M urea in the same volume, under vibration at 37 ° C for 30 minutes, and centrifuged at 12000 rpm, 4 ° C for 15 minutes to obtain the supernatant and the precipitate. The supernatant (that is, 4M urea dissolved the supernatant) was maintained; and the precipitate was additionally resuspended in 8M urea in the same volume, under vibration at 37 ° C for 30 minutes, and centrifuged, and the supernatant (ie 8M urea dissolved the supernatant) was maintained.
[109] The SDS-PAGE analytical results of the fractions obtained (Coomassie brilliant blue staining was used for visualization, the same below, see the methods in “The Molecular cloning Experiment Guide”, 2nd Edition) was shown in Figure 2. The results showed that fusion proteins were expressed in inclusion bodies (see figure 2A), and CRM197-L-E2s, 389-L-E2, AL-E2, and A-E2s, were mainly dissolved in 4M urea (see figure 2B), CRM197-L-E2s, 389-L-E2s, AL-E2s, and 389-E2s were mainly dissolved in 8M urea (see figure 2C). Supernatants dissolved in 4M urea or supernatants dissolved in 8M urea, containing the fusion protein, were dialyzed to PBS, respectively, to achieve the fusion proteins with a purity of about 80% (see, figure 2D) .
[110] Purification of the A-L-E2 fusion protein by anion exchange chromatography. Sample: a solution of protein A-L-E2 with a purity of about 80% as obtained above. Equipment: AKTA 100 explorer, preparatory liquid chromatography system, produced by GE Healthcare (ie the original from Amershan Pharmacia Co.). Chromatography medium: Sepharose Q fast flow (GE Healthcare Co). Column Volume: 15 mm x 20 cm Buffer: 20 mM phosphate buffer, pH 7.7 + 4M urea 20mM phosphate buffer, pH 7.7 + 4M urea + 1M NaCl Flow rate: 6mL / minute Detector wavelength: 280 nm Elution protocol: elution of the protein of interest with 10 mM NaCl, elution of the unwanted protein with 300 mM NaCl, and collection of the fraction eluted with 150 mM NaCl. Purification of the A-L-E2 fusion protein by hydrophobic interaction chromatography: Equipment: AKTA 100 explorer of the preparatory liquid chromatography system, produced by GE Healthcare (ie the original by Amershan Pharmacia Co.). Chromatography medium: Sepharose Phenyl Fast Flow (GE Healthcare Co.). Column volume: 15 mm x 20 cm Buffer: 20 mM phosphate buffer, pH 7.7 + 4 M urea + 0.5 M (NH4) 2SO4. 20 mM phosphate buffer, pH 7.7 + 4M Flow rate: 5 mL / minute. Wavelength detector: 280 nm Sample: the fraction eluted with 150 mM NaCl as obtained in the previous step was dialyzed in a buffer (20 mM phosphate buffer, pH 7.7 + 4M urea + 0.5 M of ( NH4) 2SO4), and then it was used as a sample.
[111] The fraction eluted with 0.1 M and 0 M of (NH4) 2SO4) was dialysed and renatured inside the PBS, and then 10 μ l was taken by the SDS-PAGE analysis, and electrophoresis bands were visualized by staining Blue shiny coomassie. The results showed that after the above purification steps, the A-L-E2 fusion protein had a purity above 90% (see figure 3). Example 4: Analysis of the properties of the fusion proteins constructed in Example 2. Determination of the reactivity of the fusion proteins with antibodies by Western blotting.
[112] The reactivity of fusion proteins with the neutralizing HEV monoclonal antibody 8C11 (see, Zhang et al., “Vaccine”, 23 (22): 2881-2892 (2005)) and anti-CRM197 polyclonal antiserum (which was prepared by immunizing the mouse with CRM197 using methods well known in the art, and serum reactivity was confirmed by commercially available CRM197) were determined by Western blotting. The dialysed and renatured samples were transferred to the nitrocellulose membrane for blotting after SDS-PAGE separation; 5% skimmed milk was used to block the membrane for 2 hours, monoclonal antibody 8C11 diluted in a determined proportion was then added (monoclonal antibody was diluted 1: 500, and the polyclonal antiserum was diluted 1: 1000), and the reaction was carried out for 1 hour. The membrane was washed with TNT (50 mmol / L of Tris * Cl (pH 7.5), 150 mmol / L of NaCl, 0.05% Tween 20) for three times, 10 minutes for each time. Goat anti-mouse alkaline phosphatase (KPL product) was then added, the reaction was carried out for 1 hour, and the membrane was then washed with TNT for three times, 10 minutes for each time. NBT and BCIP (PROTOS products) were used for visualization. The results, as determined by Western blotting using the fusion proteins and the HEV neutralizing monoclonal antibody 8C11, were shown in Figure 4. The results showed that all the fusion proteins tested had significant reactivity with the neutralizing HEV monoclonal antibody 8C11 .
[113] Determination of the reactivity of fusion proteins with several specific HEV antibodies by ELISA.
[114] The reactivity of fusion proteins and the control proteins E2 and HEV-239 with various specific HEV antibodies (Gu Ying et al., “Chinese Journal of Virology”, 19 (3): 217223 (2003)) was determined by indirect ELISA. The dialysed and renatured samples were diluted in 1X PBS (1 μ g / ml), and then added to a 96-well microplate (Beijing Wantai Co.) with 100 μ l / well and incubated at 37 ° C for 2 hours. The coating solution was discarded, the plate was washed with PBST (PBS + 0.05% Tween-20) once, and then the blocking solution (2% gelatin, 5% Casein, 1% Proclin300 in PBS) was added and 200 μ l / well and incubated at 37 ° C for 2 hours.
[115] The blocking solution was discarded when detection was performed and the HEV monoclonal antibodies were diluted to a certain extent (when E2s and their fusion protein were detected, they were diluted to 1: 10,000; when E2 and its fusion protein were detected, they were diluted to 1: 100000; when the reactivity of AL-E2, proteins 239 and E2 were compared, monoclonal antibodies were subjected to 10 serial dilutions, with 1 mg / ml being used as the initial concentration, and the monoclonal antibody in its initial concentration was subjected to dilution in the same way) was added to 100 μ l / well. The mixture was incubated at 37 ° C for 1-2 hours. The plate was then washed with PBST for 5 times, and the HRP-labeled goat anti-mouse (KPL product) (1: 5000) was then added at 100 μl / well and incubated at 37 ° C for 30 minutes. ; the plate was then washed with PBST for five times, the HRP substrate (Beijing Wantai, Co.) was then added at 100 μl / well and incubated at 37 ° C for 15 minutes. 2M sulfuric acid was added at 50 μ l / well to stop the reaction, and the microplate reader (Sunrise Type, a Tecan Co. product) was then used to read the OD450 / 620 value. The results of the ELISA using the fusion proteins with the monoclonal antibodies were shown in Figure 5. The results showed that the reactivity of the E2s protein with the monoclonal antibody was significantly improved, after its fusion with CRM197 or a fragment thereof, where the reactivity of AL-E2s and A-E2s was significantly improved; the reactivity of protein E2 with the specific monoclonal antibody HEV was retained or improved, after its fusion with CRM197 or a fragment thereof.
[116] Analysis of the reactivity of the purified A-L-E2 fusion protein by chromatography.
[117] The reactivity of the A-L-E2 fusion protein, purified by two-step chromatography, was analyzed by indirect ELISA (see the concrete process in the previous step). The ELISA result was shown in Figure 6. The result showed that the reactivity of A-L-E2 with the specific monoclonal antibody HEV was comparable to that of the HEV-239 and E2 proteins.
[118] Sedimentation velocity (SV) analysis of the A-L-E2 fusion protein.
[119] The apparatus used in the experiment was the US Beckman XL-A analytical supercentrifuge, which was equipped with an optical detection system and An-50Ti and AnNa- 60Ti rotors. The sedimentation speed (SV) method (c (s) algorithm, see P. Schuck et al., “Biophys. J.”, 78: 16061619 (2000)) was used to analyze the sedimentation coefficient of the fusion protein AL-E2. The analytical result was shown in figure 7. The result showed that the A-L-E2 fusion protein was mainly present in a dimer form, and some dimers can be further polymerized to form a tetramer. Example 5: Analysis of the immunogenicity of the fusion proteins constructed in Example 2. Titration of the antibody induced by the fusion proteins.
[120] The mice used in the experiment were female, 6-week-old BALB / C mice. By using the aluminum adjuvant, the mice were immunized by intraperitoneal injection of the fusion proteins that were prepared by the methods in Example 3 and renatured to PBS and the control proteins HEV-239, E2 and E2s, respectively. The injection volume was 1 ml, and two dose groups (a 5 μ g dose group or a 0.5 μ g dose group) were used. Primary immunization was performed at week 0, and immunization and induction was performed at week 2 and 4.
[121] HEV-239 was used to coat a plate, and the antibody titer in the serum as induced by the fusion proteins and the control proteins, was measured by similar indirect ELISA analysis as described above. The results of detection of antibody titration in serum within 3 months after immunization were shown in figure 8. The results showed that serum conversion occurred in mouse serum at week 4 in both 5μ g- and 0 dose groups. , 5 μ g, and the antibody titer reached the highest value at week 5 or 6. In particular, in the 5 μ g dose group, the highest antibody titer was obtained when AL-E2 was used, which reached 106 in the week 6; and the antibody titer induced by the fusion protein was higher or comparable to that of the HEV-239 protein. In the 0.5 μ g dose groups, the antibody titer of the fusion proteins was significantly higher than that of HEV-239, and the antibody titre induced by the AL-E2 protein reached 106 at week 5. In addition, the serum -conversion did not occur in the immune serum when using E2 and E2s, in dose groups 5μ g- and 0.5 μ g. as seen from the results above, the immunogenicity of the constructed fusion proteins was significantly greater than that of the antigen protein (E2 and E2s) alone, indicating that the CRM197 of the invention or a fragment thereof significantly improved the immunogenicity of antigen protein fused to it, and could be used as an intramolecular adjuvant.
[122] Investigation into the median effective dose (ED50) of the A-L-E2 fusion protein.
[123] In the experiment, the immunogenicity of the fusion proteins was investigated by determining the median effective dose (ED50). The experimental animals used were female BALB / c mice with 3-4 weeks of age. A-L-E2 was mixed with the aluminum adjuvant, and the initial dose was 1 μ g / mouse, and was subjected to 1: 3 serial dilution, resulting in 8 dose groups in total. In addition, HEV-239 (HEV recombinant vaccine) was used as a control and the initial dose was 1.6 μ g / mouse, and was subjected to serial dilution in 1: 4, resulting in a total of 4 dose groups. 6 mice were used in each group. Immunization was performed through a single intraperitoneal injection.
[124] Peripheral venous blood was taken after 4 weeks after immunization, the serum was separated, and the serological conversion rate was determined by ELISA analysis as described above. When the ELISA value of 100 times the diluted serum was greater than the cutoff value (ie, three times the mean negative value), the serum was listed as positive. The median effective dose (ED50) was calculated using the Reed-Muench method.
[125] The serological conversion rate for the A-L-E2 fusion protein was shown in table 2, and the serological conversion rate for the HEV-239 vaccine was shown in table 3. Table 2:
Table 3:

[126] The results showed that the ED50 of HEV-239 was 11 times that of AL-E2, indicating that CRM197 of the invention or a fragment thereof significantly improved the antigen protein fused to it, and could be used as intramolecular adjuvant. For the time being, since the immunogenicity of the AL-E2 fusion protein was significantly higher than that of the HEV-239 vaccine in the form of a viral-type particle, the fusion protein can be used for the preparation of a new vaccine that is more effective for Hepatitis E. Example 6:
[127] Design and cloning of fusion proteins comprising CRM197 or a fragment thereof and an M2e protein from the influenza virus.
[128] In the Example, expression vectors of the fusion proteins were exemplarily constructed. The clone design of the exemplary fusion proteins constructed is shown in figure 9, with the fusion proteins each comprising CRM197 or a fragment thereof and an M2e protein from the influenza virus, optionally using a linker. Fusion protein clone. M2e fused to the CRM197 C-terminal or a fragment thereof.
[129] The amplification product (i.e., the full length gene from CRM197) obtained in example 1 was used as a template. The previous primer was CRM197F (SEQ ID NO: 45), at the 5 'terminal from which the CAT ATG restriction endonuclease NdeI site was introduced, with ATG being the start codon in the E. coli system. The reverse primer was CRM197-linker R1 (SEQ ID NO; 46), 389-linker R1 (SEQ ID NO: 47) and an A-linker R1 (SEQ ID NO: 48), respectively, at the 5 'end at which BamHI site of the GGA TCC restriction endonuclease was introduced. The PCR reaction was performed in a PCR thermocycler (Biometra T3) under the following conditions. The sequences of the primers used are shown in Table 4.

[130] The amplification products were DNA fragments of about 1600 bp, 1200 bp and 600 bp in length, respectively.
[131] In addition, the plasmid PHW2000 (stored in our laboratory, comprising the full-length M2 gene) was used as a template. The previous primer was M2eF1 (SEQ ID NO: 49), at the 5 'end from which the GAM TCC restriction endonuclease BamHI site was introduced. The reverse primer was M2eR (SEQ ID NO: 50), at the 5 'terminal from which the GAA TTCC restriction endonuclease site EcoRi was introduced. The PCR reaction was performed in a PCR thermocycler (Biometra T3) under the following conditions. The sequences of the primers used were shown in Table 4.

[132] The amplification products were fragments of DNA about 70bp in length, respectively.
[133] The amplification products as obtained above were linked within the commercially available pMD 18-T vector (produced by TAKARA Co.), respectively, and designated as pMD 18-T-CRM197-L1, pMD 18-T-389- L1 and pMD 18-TA-L1 as well as pMD 18-T-M2e. As identified by the cleavage enzymes NdeI / BamHI and BamHi / jEcoRI, positive clones pMD 18-T-CRM197-L1, pMD-18-T-389-L1, pMD 18-TA-L1 and pMD 18-T- respectively M2e were obtained.
[134] As confirmed by the M13 (+) primer, the correct nucleotide sequences of interest were inserted into the obtained plasmids pMD 18-T-CRM197-L1, pMD 18-T-389-L1; pMD 18-T-A-L1 and pMD 18-T-M2e.
[135] Plasmids pMD 18-T-CRM197-L1, pMD 18-T-389-L1 and pMD 18-T-A-L1 were digested by the enzyme NdeI / BamHI. The fragments obtained by the cleavage enzyme were ligated into the prokaryotic expression vector pTO-T7 digested by the enzyme NdeI / BamHI (Luo Wenxin et al., “Chinese Journal of Biotechology”, 2000, 16: 53-57), and were transformed in E.coli ER2566 (purchased from Invitrogen Co.), after extraction of the plasmids, as indicated by the cleavage enzyme NdeI / BamHI, the positive plasmid pTO-T7-CRM197-L1, pTO-T7-389-L1 and pTO-T7 -A-L1, in which the CRM197-Li, 389-L1 and A-L1 fragments were inserted, respectively, were obtained.
[136] pTO-T7-CRM197-L1, pTO-T7-389-L1, pTO-T7-A-L1 and pMD-18-T-M2e were digested by the enzyme BamHI / EcoRi. The M2e fragment obtained was ligated into the vectors pTO-T7-CRM197-L1, pTO-T7-389-l1 and pTO-T7-A-l1 digested by the enzyme BamHI / EcoRi, respectively. As identified by the NdeI / EcoRI cleavage enzyme, the positive expression vectors pTO-T7-CRM197-LM2e, pTO-T7-389-L-M2e, and pTO-T7-AL-M2e, within which CRM197-L- M2e (SEQ ID NO: 33, 34), 389-L-M2e (SEQ ID NO: 35, 36), or AL-M2e (SEQ ID NO: 37, 38) was inserted, respectively, were obtained.
[137] M2e fused to the CRM197 N-terminal or a fragment thereof. Plasmid PHW2000 (stored in our laboratory, containing the full-length M2 gene) was used as a template. The previous primer was M2eF2 (SEQ ID NO: 51), at the 5 'terminal from which the CAT ATG restriction endonuclease NdeI site was introduced, with ATG being the start codon in the E. coli system. The reverse primer was M2e-ligand R (SEQ ID NO: 52), at the 5 'terminal from which the BamHI of the GGA TCC restriction endonuclease was introduced. The PCR reaction was performed in a PCR thermocycler (Biometra T3) under the following conditions.

[138] The amplification products were fragments of DNA about 100 bp in length.
[139] In addition, the amplification product (i.e., the full length gene from CRM197) obtained in Example 1 was used as a template. The previous primer was CRM197F2 (SEQ ID NO: 53), at the 5 'end from which the restriction endonuclease BamHI GGA TCC was introduced. The reverse primer was CRM197 R2 (SEQ ID NO: 54), 389 R (SEQ ID NO: 55), and AR (SEQ ID NO: 56), at the 5 'terminal on which the GARI TTC restriction endonuclease EcoRI site was introduced. The PCR reaction was performed in a PCR thermocycler (Biometra T3) under the following conditions. The sequences of the primers used are shown in Table 4.

[140] The amplification products were fragments of DNA of about 1600bp, 1200 bp and 600bp in length, respectively.
[141] The amplification products as obtained above were linked within the commercially available pMD 18-T vector (produced by TAKARA Co.), respectively, and designated as pMD 18-T-M2e-L as well as pMD 18-T-CRM197 , pMD 18-T-389 and pMD 18-TA, respectively. As identified by the cleavage enzyme NdeI / BamHI and BamHI / EcoRI, respectively, positive clones pMD 18-T-CRM197, pMD 18-T-389, pMD 18-T-A, and pMD 18-T-M2e-L were obtained.
[142] As confirmed by the M13 (+) primer, the correct nucleotide sequences of interest were inserted into the obtained plasmids pMD 18-T-CRM197, pMD 18-T-389, pMD 18-TA, and pMD 18-T- M2e, respectively.
[143] The plasmid pMD 18-T-M2e-L was digested by the enzyme NdeI / BamHI. The fragments obtained by the cleavage enzyme were then ligated into the prokaryotic expression vector pTO-T7 digested by the enzyme NdeI / BamHI (Luo Wenxin et al., “Chinese Journal of Biotechnology”, 2000, 16: 53-57), and was transformed into E. coli ER 2566 (purchased from Invitrogen Co.), after extraction of the plasmids, as indicated by the cleavage enzyme NdeI / BamHI, the positive plasmid pTO-T7-M2e-L, within which the M2e-L fragment was inserted, it was obtained.
[144] pTO-T7-M2e-L, pMD 18-T-CRM197, pMD 18-T-389 and pMD 18-T-A were digested by the enzyme BamHI / EcoRi. The obtained fragments CRM197, 389 and A were ligated into the vector pTO-T7-M2e-L digested by the enzyme BamHi / EcoRi, respectively. As indicated by the NdeI / EcoRI cleavage enzyme, the positive expression vectors pTO-T7-M2e-L-CRM197, pTO-T7-M2e-L-389, and pTO-T7-M2e-LA, within which M2e-L -CRM197 (SEQ ID NO: 39, 40), M2e-L-389 (SEQ ID NO: 41, 42), and M2e-LA (SEQ ID NO: 43, 44) were inserted respectively, were obtained.
[145] The primer sequences used in the Example are listed in Table 4. Table 4:


[146] 1μL of plasmids pTO-T7-CRM197-L-M2e, pTO-T7-389-L-M2e, pTO-T7-AL-M2e, pTO-T7-M2e-L-CRM197, pTO-T7-M2e- L-389 and pTO-T7-M2e-LA (0.15mg / ml) were separately used to transform 40 μL of competent E. coli ER 2566 (purchased from Invitrogen) prepared by the calcium chloride method, and then the bacteria were plated in solid LB medium (the components of the LB medium: 10 g / L of peptone, 5 g / L of yeast powder, and 10 g / L of NaCl, the same below) containing kanamycin (in a final concentration of 100 mg / ml, the same below). The plates were statically incubated at 37 ° C for about 10-12 hours until the individual colonies could be seen clearly. The individual colonies of the plates were transferred to a tube containing 4 ml of liquid LB medium containing kanamycin. The cultures were incubated in a shaking incubator at 180 rpm for 10 hours at 37 ° C, and then 1 ml of the bacterial solution was taken and stored at -70 ° C. Example 7:
[147] The expression, isolation and renaturation of the fusion proteins constructed in Example 6.
[148] 5 μl of the bacterial solution, taken from an ultra low temperature in the -70 ° C freezer, was seeded in 5 ml of liquid LB medium containing kanamycin, and then it was grown at 37 ° C, 180 rpm under stirring until OD600 reaches about 0.5. The resulting solution was transferred to 500 ml of LB medium containing kanamycin, and then it was grown at 37 ° C, 180 rpm with stirring for 4-5 hours. When OD600 reached about 1.5, IPTG was added to a final concentration of 0.4 mM, and the bacteria were induced under stirring at 37 ° C for 4 hours.
[149] After incubation, centrifugation was performed at 8000g for 5 minutes to collect the bacteria, and then the bacterium was resuspended in a lysis solution in a ratio of 1g of bacteria to 10 ml of the lysis solution (20 mM of buffer) Tris, pH 7.2; 300 mM NaCl), in an ice bath. The bacterium was treated with a sonicator (Sonics VCX750 Type Sonicator) (Conditions: operating time 15 minutes, pulse 2 seconds, intermissions 4 seconds, output energy 55%). The bacterial lysate was centrifuged at 12000 rpm, 4 ° C for 5 minutes (the same below), and the supernatant and the precipitate (that is, inclusion body) after breaking the bacteria by ultrasonication were collected, respectively. 2% Triton-100 of the same volume was used to wash the precipitate, the resulting mixture was vibrated for 30 minutes, centrifuged, and the supernatant was discarded. The precipitate was resuspended in buffer I (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM EDTA), under vibration for 30 minutes, centrifuged, and the supernatant was discarded. The precipitate was then resuspended in 2M urea, under vibration at 37 ° for 30 minutes, centrifuged, and the supernatant and precipitate were obtained. The supernatant was maintained; and the precipitate was resuspended in 4M urea in the same volume, under vibration at 37 ° C for 30 minutes, and centrifuged at 12000 rpm, 4 ° C for 15 minutes to obtain the supernatant and the precipitate. The supernatant (ie, supernatant dissolved in 4M urea) was maintained; and the precipitate was additionally resuspended with 8M urea in the same volume; under vibration at 37 ° C for 30 minutes, and centrifuged, and the supernatant (ie, the supernatant dissolved in 8M urea) was maintained.
[150] The fractions obtained were analyzed by SDS-PAGE (Coomassie brilliant blue staining was used for visualization, the same below, see the methods in “The Molecular Cloning Experiment Guide”, 2nd edition). The results showed that the fusion proteins were expressed in inclusion bodies (see figures 10A and 10B), CRM197-L-M2e, 389-L-M2e, M2e-L-CRM197 and M2e-L-389 were mainly dissolved in 8M urea, and AL-M2e and M2e-LA were mainly dissolved in 4M urea. The supernatants dissolved in 4M urea containing AL-M2e or M2e-LA or the supernatant dissolved in 8M urea containing CRM197-L-M2e, 389-L-M2e, M2e-L-CRM197 or M2e-L-389, were dialyzed in PBS, respectively, to achieve the fusion protein with a purity of about 80% (see figures 10C-10F). Example 8: Analysis of the properties of the fusion proteins constructed in Example 6. Determination of the reactivity of the fusion proteins with antibodies by Western Blotting.
[151] The reactivity of the fusion proteins with the influenza virus 5D1 monoclonal antibody M2e and the CRM197 monoclonal antibody 1E6 (prepared in the laboratory) were determined by Western blotting. The dialyzed and renatured samples were transferred to the nitrocellulose membrane for blotting after separation by SDS-PAGE; 5% skimmed milk was used to block the membrane for 2 hours, and then the monoclonal antibody 5D1 diluted in 1: 500 was added. The reaction was carried out for 1 hour. The membrane was then washed with TNT (50 mmol / L of Tris * Cl (pH 7.5), 150 mmol / L of NaCl, 0.05% Tween 20) for three times, 10 minutes each time. Goat anti-mouse alkaline phosphatase (KPL product) was then added. The reaction was carried out for 1 hour, and the membrane was then washed with TNT three times, 10 minutes each time. NBT and BCIP (PROTOS product) were used for visualization. The results, as determined by Western blotting using the fusion proteins and the influenza virus M2e monoclonal antibody (figures 11A - 11D) or the CRM197 monoclonal antibody 1E6 (Figures 11E-11H), were shown in figure 11. The results showed that all fusion proteins had significant reactivity with the influenza virus M2e specific monoclonal antibody 5D1 and the CRM197 specific monoclonal antibody 1E6.
[152] Determination of the reactivity of fusion proteins with several M2e specific monoclonal antibodies and the specific CRM197 antibody by ELISA.
[153] The reactivity of the fusion proteins and the control protein GST-M2e with several specific M2e antibodies and specific monoclonal antibody CRM197 1E6 (the antibodies used in the experiment are known in the art, or commercially available or prepared in the laboratory) was determined by EILSA indirect. For example, the O19 antibody was a protective antibody against influenza known in the art (see, Fu et al., "Virology", 2009, 385: 218-226). The dialysed and renatured samples were diluted in 1X PBS (1μ g / ml), and then added in 96-well microplates (Beijing Wantai Co.), in 100 ml / well and incubated at 37 ° C for 2 hours. The coating solution was discarded, the plate was washed with PBST (PBS + 0.05% Tween-20) once, and then the blocking solution (2% gelatin, 5% casein, 1% Proclin 300 , in PBS) was added at 180μ l / well and incubated at 37 ° C for 2 hours. The blocking solution was discarded when detection was performed, and the anti-M2e antibody or the CRM197 antibody diluted to a certain extent (0.002 mg / ml was used as the initial concentration for 2 times the gradient dilution) was added in 100 μ l / well. The mixture was incubated at 37 ° C for 1 hour. The plate was washed with PBST for five times, the HRP-labeled goat anti-mouse (KPL product) (1: 5000) was then added at 100 μl / well and incubated at 37 ° C for 30 minutes. The plate was washed with PBST five times, the HRP substrate (Beijing Wantai Co.) was then added at 100 μl / well and incubated at 37 ° C for 15 minutes. 2m sulfuric acid was added at 50 μl / well to stop the reaction, and the microplate reader (Sunrise Type, a Tecan Co. product) was then used to read the OD450 / 620 value. The ELISA results using the antibody fusion proteins were shown in Figures 12A and 12B. The results showed that when compared to the M2e protein alone, the reactivity of the M2e protein with several anti-M2e specific monoclonal antibodies was maintained or improved after its fusion with CRM197 or a fragment thereof. Analysis of the sedimentation speed (SV) of the fusion proteins.
[154] The apparatus used in the experiment was the US Beckman XL-A analytical supercentrifuge, which was equipped with an optical detection system and An-50Ti and An- 60Ti rotors. The sedimentation speed (SV) method (c (s) algorithm, see P. Schuck et al., “Biophys. J.”, 78: 16061619 (2000)) was used to analyze the sedimentation coefficient of the fusion protein . The analytical results are shown in figures 13A - 13F. The results showed that among the fusion proteins constructed in Example 6, A-L-M2e and M2e-L-A were mainly present in the form of monomer and tetramer; and 389-L-M2e was mainly present in the form of dimers and polymers; M2e-L-389 was mainly present in the form of monomer and polymer, CRM197-L-M2e was mainly present in the form of dimer and polymer; and M2e-L-CRM197 was mainly present in the form of monomer and polymer. Example 9: Analysis of the immunogenicity of the fusion proteins constructed in Example 6.
[155] The mice used in the experiment were female, 6-week-old BALB / C mice. By using the aluminum adjuvant, the mice were immunized by intraperitoneal injection of the fusion proteins that were prepared by the methods in Example 6 and renatured to PBS and the GST-M2e control proteins, respectively. The injection volume was 1ml, and two dose groups (a 5μg dose group or a 0.5 μg dose group) were used. Primary immunization was performed at week 0, and induction immunization was performed at weeks 2 and 4.
[156] GST-M2e was used to coat a plate, and the antibody titer in the serum as induced by the fusion proteins and the control proteins, was measured by indirect ELISA analysis similar to that described above. The results of detection of antibody titration in serum within 4 months after immunization are shown in figures 14A and 14B. The results showed that after the second induction immunization, the immunogenicity of the constructed fusion proteins was significantly greater than that of the antigen protein (GST-M2e) alone, indicating that the CRM197 of the invention or a fragment thereof (no matter being located at the N-terminus or the C-terminus of the fusion protein) significantly improved the immunogenicity of the antigen protein fused to it, and could be used as an intramolecular adjuvant.
[157] Although specific embodiments of the invention have been described in detail, those skilled in the art would understand that, according to the teachings described in the report, various modifications and changes can be made without departing from the spirit and scope of protection of the invention, as generally described and that such modifications and changes are within the spirit and scope of protection of the invention. The scope of the present invention is given by the appended claims and any equivalents thereof.

权利要求:
Claims (23)
[0001]
1. Fusion protein, characterized by the fact that it comprises a fragment of CRM197 and a target protein, being that said fragment of CRM197 increases the immunogenicity of the target protein, being that said fragment of CRM197 consists of the amino acids (aa) of 1-190 or aa from 1-389 of SEQ ID NO: 2.
[0002]
2. Fusion protein according to claim 1, characterized in that the target protein is an immunogenic fragment of the same or a HEV capsid protein, the HEV capsid protein having an amino acid sequence as represented by SEQ ID NO: 31;
[0003]
3. Fusion protein according to claim 2, characterized in that the immunogenic fragment of the HEV capsid protein comprises or is HEV-239 (aa 368606 of the HEV capsid protein), E2 (aa 394-606 of the HEV capsid) or E2s (aa 455-606 HEV capsid protein).
[0004]
4. Fusion protein, according to claim 1, characterized in that the target protein is an M2 protein of the influenza virus or an immunogenic fragment thereof, with the M2 protein of the influenza virus having an amino acid sequence as represented in SEQ ID NO: 32.
[0005]
Fusion protein according to claim 4, characterized in that the immunogenic fragment of the M2 protein comprises or is M2e (aa 1-24 of the M2 protein).
[0006]
6. Fusion protein according to any one of claims 1 to 5, characterized in that in the fusion protein, said fragment of CRM197 is linked to an N-terminal and / or C-terminal of the target protein, with or without a binder.
[0007]
7. Fusion protein, according to claim 1, characterized in that the fusion protein comprises said fragment of CRM197 and a HEV capsid protein or an immunogenic fragment thereof; which are linked together, with or without a linker, the HEV capsid protein having an amino acid sequence as represented in SEQ ID NO: 31.
[0008]
8. Fusion protein according to claim 7, characterized in that the fusion protein has a protein having an amino acid sequence as represented in SEQ ID NOs: 8, 10, 12, 14, 16 or 18.
[0009]
9. Fusion protein according to claim 1, characterized in that the fusion protein comprises said fragment of CRM197 and an M2 protein from the influenza virus or an immunogenic fragment thereof, which are linked together, with or without a ligand , with the M2 protein of the influenza virus having an amino acid sequence as represented in SEQ ID NO: 32.
[0010]
10. Fusion protein according to claim 9, characterized in that the fusion protein has an amino acid sequence as shown in SEQ ID NOs.:36, 38, 40, 42 or 44.
[0011]
11. Polynucleotide, characterized by the fact that it encodes the fusion protein as defined in any one of claims 1 to 10.
[0012]
12. Construct, characterized by the fact that it comprises polynucleotide, as defined in claim 11.
[0013]
13. Vector, characterized by the fact that it comprises the polynucleotide, as defined in claim 11 or the construct, as defined in claim 12.
[0014]
14. Host cell, characterized by the fact that it comprises the polynucleotide as defined in claim 11, the construct as defined in claim 12, or the vector as defined in claim 13, the host cell being a prokaryotic cell.
[0015]
15. Pharmaceutical composition or vaccine, characterized in that it comprises the fusion protein as defined in any one of claims 1 to 10, and a pharmaceutically acceptable carrier and / or excipient.
[0016]
16. Use of the fusion protein, as defined in any of claims 2 to 3 and 7 to 8, characterized by the fact that it is directed to the manufacture of a pharmaceutical composition for the prevention and / or treatment of hepatitis E.
[0017]
17. Use of the fusion protein, as defined in any of claims 4-5 and 9-10, characterized by the fact that it is for the production of a pharmaceutical composition to prevent and / or treat influenza or influenza virus infection.
[0018]
18. Method for modifying a target protein to improve its immunogenicity, characterized by the fact that it comprises obtaining a fusion protein containing the CRM197 fragment as defined in claim 1 and the target protein, in order to improve the immunogenicity of the target protein.
[0019]
19. Method according to claim 18, characterized in that the fusion protein is obtained by expressing the fusion of the CRM197 fragment with the target protein, with or without using a linker.
[0020]
20. Method according to either of claims 18 or 19, characterized in that the target protein is a HEV capsid protein, an M2 protein from the influenza virus, or an immunogenic fragment thereof, the HEV capsid protein being have an amino acid sequence as shown in SEQ ID NO: 31, and the influenza virus M2 protein have an amino acid sequence as shown in SEQ ID NO: 32.
[0021]
21. Use of the CRM197 fragment, as defined in claim 1, in improving the immunogenicity of a target protein, characterized in that it obtains a fusion protein comprising the CRM197 fragment and the target protein.
[0022]
22. Use of the fragment, according to claim 21, characterized in that the fusion protein is obtained by expressing the fusion of the CRM197 fragment with the target protein, with or without the use of a ligand.
[0023]
23. Use according to either of claims 21 or 22, characterized in that the target protein is a HEV capsid protein, an M2 protein from the influenza virus, or an immunogenic fragment thereof, the HEV capsid protein being has an amino acid sequence as represented by SEQ ID NO: 31, and the influenza virus M2 protein has an amino acid sequence as represented in SEQ ID NO: 32.

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同族专利:

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MX2013014109A|2014-06-05|

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law|

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: A61K 39/39 (2006.01), A61K 39/05 (2006.01), A61K 3 |

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-03-19| B07E| Notice of approval relating to section 229 industrial property law|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-10-29| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|

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2020-03-17| B07A| Technical examination (opinion): publication of technical examination (opinion)|

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2020-09-08| B09A| Decision: intention to grant|

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2020-12-01| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 01/06/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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CN201110145419|2011-06-01|

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CN201110145419.2|2011-06-01|

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PCT/CN2012/076378|WO2012163289A1|2011-06-01|2012-06-01|Fusion protein comprising diphtheria toxin non-toxic mutant crm197 or fragment thereof|

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