• 专利附录
  • 专利说明
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    • 专利摘要:
    Use of the ifnar protein as an antiviral. The present invention relates to the use of the ifnar protein in the manufacture of a medicament for the prevention, control, treatment and/or alleviation of a viral disease. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2626002A1
    申请号:ES201531862
    申请日:2015-12-21
    公开日:2017-07-21
    发明作者:Óscar FERNÁNDEZ FERNÁNDEZ；Begoña OLIVER MARTOS；Teresa ÓRPEZ ZAFRA；Laura LEYVA FERNÁNDEZ；José PAVÍA MOLINA
    申请人:Universidad de Malaga；Servicio Andaluz de Salud；
    IPC主号:
    专利说明:

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    Use of IFNAR protein as an antiviral FIELD OF THE INVENTION
    The present invention is within the field of biomedicine and biotechnology, and refers to the use of the isolated IFNAR2.3 soluble receptor, produced recombinantly, in the prevention, control, treatment and / or relief of a viral disease. BACKGROUND OF THE INVENTION
    10 The advances produced in the therapy of viral diseases are of lesser magnitude than those that have been achieved for the treatment of bacterial infections. The development of highly effective broad spectrum antiviral agents is a primary objective shared by the fields of virology and pharmacology.
    15 Viruses are intracellular parasites that use the metabolic machinery of the infected host cell. Therefore, the development of antiviral drugs presents a series of difficulties associated with said mandatory parasitic character. It is difficult to achieve adequate antiviral activity without affecting the metabolism of the host cell and without
    20 cause negative effects on other uninfected cells of the organism.
    Current viral infectivity control strategies are based on the identification of agents capable of intervening in the essential steps of viral infection, such as entry (fusion, endocytosis), replication, assembly, as well as drugs directed against the
    25 viral envelope. Another strategy would be aimed at modulating the cellular defense system.
    The identification of broad-spectrum antiviral agents focused on decreasing viral infectivity or modulating host defenses would be an important contribution in the field of virology for the improvement of human health and control
    30 viral epidemics.
    IFNß exerts its biological activity through interaction with the IFNAR surface receptor formed by two subunits, IFNAR1 and IFNAR2. Following the binding of IFNβ to IFNAR2, dimerization of the two subunits and activation of the cascade of
    35 intracellular signaling whose signal is transduced to the nucleus through the Jak-Stat pathway. In this way, the antiviral, antiproliferative and immunomodulatory activities of IFNß are exercised.
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    The IFNAR2 subunit of the recipient undergoes alternative mRNA processing that gives
    5 place to three different isoforms: a short isoform (IFNAR2b), a functionally active long isoform (IFNAR2c) and the soluble isoform (sIFNAR2, IFNAR2.3 or IFNAR2a). Only IFNAR2c acts as a functional receptor together with IFNAR1 and is able to mediate the biological effects of IFNß. sIFNAR2 that lacks cytoplasmic and transmembrane domains, has been identified in human biological fluids and although its role has not
    10 is defined, it has been suggested that it may have neutralizing ability of IFNβ binding with the IFNAR2 receptor. In this way you could exercise modulating functions depending on the concentration you are in; on the one hand it could neutralize the binding of IFNβ to the IFNAR receptor or, on the contrary, prolong the half-life of circulating IFNß, preventing its degradation or the formation of oligomers. The function remains unknown today
    15 of the soluble variant of IFNAR2.
    The inventors of the present invention have proven the antiviral effect of a recombinant sIFNAR2 protein, analogous to the soluble isoform of the IFNβ receptor. 20 BRIEF DESCRIPTION OF THE INVENTION
    A first aspect of the invention relates to the use of a recombinant protein, hereinafter recombinant protein of the invention, obtainable by a method comprising:
    A) integrating an insert with the nucleotide sequence SEQ ID NO: 1 into a genetic construct or an expression vector, b) transforming a host with the expression vector of step (a), c) inducing the expression of the recombinant protein , d) extract the recombinant protein, and
    30 e) purify the recombinant protein, in the preparation of a medicament for the prevention, improvement, treatment and / or relief of a viral disease.
    In a preferred embodiment of this aspect of the invention, the expression vector is the prelinearized vector pEcoli-Cterm 6xHN Linear.
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    In another preferred embodiment of this aspect of the invention, the host of step (b) are expression bacteria. Preferably, the expression bacteria are E.coli BL21 (DE) bacteria.
    In another preferred embodiment of this aspect of the invention, the integration of the nucleotide sequence SEQ ID NO: 1 of step (a) is performed by a ligation process.
    In another preferred embodiment of the invention, a lyophilisate comprising the In-Fusion enzyme is used in the ligation.
    10
    In another preferred embodiment, the insert was synthesized using nucleotide sequence primers SEQ ID NO: 3 and SEQ ID NO: 4.
    Another aspect of the invention relates to the use of an antibody or a fragment of the
    The same, hereinafter antibody of the invention, which specifically recognizes the recombinant protein of the invention, in the preparation of a medicament for the prevention, control, treatment and / or relief of a viral disease.
    In a preferred embodiment of this aspect of the invention, where the antibody is obtained by injection of the recombinant protein of the invention into an appropriate animal, and collected, and optionally by purification of animal antisera.
    In another preferred embodiment of this aspect of the invention, the antibody is a monoclonal antibody.
    25
    Another aspect of the invention relates to the use of a composition, hereafter
    composition of the invention, comprising: a) a protein comprising the amino acid sequence SEQ ID NO: 2, b) the recombinant protein of the invention,
    C) the protein of the invention (soluble IFNAR2, sIFNAR2 or IFNAR2.3) obtained by other non-recombinant means, and / or
    d) the antibody, or a fragment thereof, of the invention, in the preparation of a medicament for the prevention, improvement, treatment and / or relief of a viral disease.
    35
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    In a preferred embodiment of this aspect of the invention, the composition is a pharmaceutical composition that optionally further comprises a pharmaceutically acceptable carrier and / or pharmaceutically acceptable excipients.
    In another preferred embodiment of this aspect of the invention, the composition of the invention further comprises another active ingredient. Preferably, this active ingredient is selected from the list consisting of other antivirals, analgesics, antipyretics, decongestants or other active ingredients used in the treatment of viral diseases.
    In another preferred embodiment of this aspect of the invention, the viral disease is generated by a virus that is selected from the list consisting of: hepatitis A virus (HAV), hepatitis B virus (HBV), virus hepatitis C (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), hepatitis F or G virus (HBV), human immunodeficiency virus (HIV), respiratory syncytial virus, virus of the flu virus
    15 encephalomyocarditis, vesicular stomatitis virus or any combination thereof. DESCRIPTION OF THE FIGURES
    Figure 1. Representative bioassay performed in the presence of IFNAR2.3. Higher intensity
    20 in the violet color, it corresponds to greater cell survival after infection of the cell culture with the virus. Figure 2. Representation of the Absorbance values obtained in the previous Bioassay in each of the conditions. It can be seen how IFNAR2.3 protects cells from virus action, with absorbance values being very close to cell control
    25 and well above viral control. Figure 3. Bar chart representing the absorbance values obtained with the three concentrations of sIFNAR2 tested. The results represent the average of the 8 replicas that have been made for each of the concentrations. It can be seen how sIFNAR2 protects cells from the action of the virus, leaving the values of
    30 absorbance well above the viral control in a statistically significant way (p = 0.012 in all comparisons). Figure 4. Second Bioassay plate performed in the presence of sIFNAR2, showing its antiviral capacity. In addition, the bioassay has been performed in the presence of B18R, which is an IFNβ inhibitor. It is shown that B18R blocks the action of IFNß but does not block the
    The action of the recombinant sIFNAR2 protein is observed as the cells remain protected in the presence of B18R + sIFNAR2.
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    Figure 5. Bar chart representing the abscesses obtained in the bioassay of Figure 4, of the viral control (CV), cell control (CC), IFN40 and IFN80. The absorbances obtained for IFNβ and sIFNAR2 in the presence of B18R are also represented. It can be seen how B18R inhibits the action of IFNβ and the cells are not protected.
    5 However, B18R does not inhibit the antiviral ability of recombinant sIFNAR2, the absorbance remaining above the CV. Figure 6. Graph representing the absorbances of the bioassay of Figure 4 as a function of the different dilutions obtained in the presence of IFNβ alone, of the combination of IFNß + sIFNAR2 and sIFNAR2 per se. It is shown that recombinant sIFNAR2 protects the
    10 cell culture in the same way as IFNβ. On the other hand, it can be observed that in most dilutions, the combination of both molecules protects cell culture more, obtaining a higher absorbance. sIFNAR2 seems to enhance the effect of IFNß and vice versa. Figure 7. Working scheme of cloning, production and purification of the protein
    15 recombinant. Figure 8. Structure of the pEcoli-Cterm 6xHN Linear vector. Figure 9. Structure of structures flanking the insert. Figure 10. Agarose gel electrophoresis of the amplified obtained by PCR. Figure 11. Alignments of nucleotide sequences in the 5´-3´ direction. In the first
    The nucleotide sequence of IFNAR2.3 is shown in the second and third lines of the nucleotide sequences with the flanking primers of the T7UP and terminal T7 insert, obtained after the plasmid sequencing process. Figure 12. Identification of the sIFNAR2 protein by MALDI-OF
    25 DETAILED DESCRIPTION OF THE INVENTION
    USE OF THE RECOMBINANT PROTEIN OF THE INVENTION
    The authors of the present invention have studied the effects of the use of a recombinant sIFNAR2 protein in the prevention, control, treatment and / or relief of a viral disease.
    Specifically, the sIFNAR2 protein, cloned and purified, has been used. In addition, by means of the cloning method employed, a histidine-asparagine tail has been added at the carboxy terminal end, the recombinant protein being fused as a
    image6
    label. After the production of the recombinant protein in the host cell, the cell lysate is passed through an affinity column for purification. The fusion protein with the tag is retained in the column while the other proteins and other contaminants are inflated through it.
    5
    Therefore, a first aspect of the invention relates to the use of a recombinant protein, hereinafter recombinant protein of the invention, obtainable by a method comprising:
    a) integrate an insert with the nucleotide sequence SEQ ID NO: 1 into a
    10 genetic construction or an expression vector, b) transforming a host with the expression vector of step (a), c) inducing the expression of the recombinant protein, d) extracting the recombinant protein, and e) purifying the recombinant protein,
    15 in the development of a medicine for the prevention, control, treatment and / or relief of a viral disease.
    The design of the vector based on genetic engineering techniques and the choice of the host cell largely determine the characteristics of the recombinant protein.
    The gene construction of section a) may comprise, in addition to the nucleotide sequence SEQ ID NO: 1, elements that regulate the expression of said sequence. Such regulatory elements include promoters and enhancers. Promoters are typically positioned in a 5 ’position with respect to the transcription initiation site
    25 or translation. Enhancers are capable of influencing gene expression when they are in a 5 ’or 3’ position with respect to the cDNA or when they are part of an intron. Regulatory sequences include, in addition to promoters, sequences that facilitate translation, intron processing signals, termination codons, signal sequences, internal ribosome binding sites (IRES), and
    30 polyadenylation.
    The expression vector of section a) comprising the nucleotide sequence SEQ ID NO: 1 or the gene construct of section a), is operatively coupled with a sequence that regulates the expression of said nucleotide sequence SEQ ID NO: 1 or of said
    35 gene construction. The person skilled in the art will note that the type of vector suitable for the expression of nucleic acids and gene constructs of the invention will depend on the organism in which it is desired to express the polynucleotide of the invention.
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    In a preferred embodiment of this aspect of the invention, the expression vector is the prelinearized vector pEcoli-Cterm 6xHN Linear.
    In another preferred embodiment of this aspect of the invention the cloning of the protein is
    performed on baculovirus and / or the vectors used are selected, but not limited to, from the
    list consisting of: pAcP (+) IE1-1, pAcP (+) IE1-2, pAcP (+) IE1-3, pAcP (+) IE1-4, 10 pAcP (+) IE1-5, pAcP (+) IE1-6, pAcUW31, pBAC-1, pBAC-2cp, pBAC-3, pBAC4x-1, pBAC-7,
    pBAC-8, pBAC-9, pBAC-10, pBacPAK8, pBacPAK9, pBACsurf-1, pBlueBac4.5,
    pBlueBacHis2, pFastBac1, pFastBac HT, pFastBac DUAL, pMbac, pMelBac, pPbac, pTriEx
    1, pVL1392 and pVL1393. Preferably, the promoters used in these vectors are:
    ie1 promoter in the case of pAcP (+) IE1-1, pAcP (+) IE1-2, pAcP (+) IE1-3, pAcP (+) IE1-4, 15 pAcP (+) IE1-5, pAcP (+ ) IE1-6; polh promoter in the case of pAcUW31, pBAC-1, pBAC-2cp,
    pBAC-3, pBAC4x-1, pBAC-7, pBAC-8, pBAC-9, pBAC-10, pBacPAK8, pBacPAK9,
    pBACsurf-1, pBlueBac4.5, pBlueBacHis2, pFastBac1, pFastBac HT, pFastBac DUAL,
    pMbac, pMelBac, pPbac, pVL1392 and pVL1393; the p10 promoter pAcUW31, pBAC4x-1,
    pFastBac DUAL, pMbac, pMelBac, pPbac and pTriEx-1. Preferably, the selection of the 20 clones is effected by antibiotic resistance genes introduced into the vector,
    more preferably resistance to ampicillin and / or gentamicin.
    A cell or host organism may comprise the gene construct or a
    vector, as defined in the invention. In principle, any type of host organism known to the person skilled in the art can be used herein.
    invention, such as a bacterial strain (Escherichia coli, Bacillus subtilis and the like),
    a strain of yeast (Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis,
    Hansenula polymorpha and the like), a transgenic plant (dicotiledoneas or
    monocotyldoneas), an insect cell, for example, baculovirus, a mammalian cell 30 (COS, CHO, C127, HeLa cells and the like) and a non-human transgenic (for example, a
    mouse, a cow, a goat, a rabbit, a pig, etc.).
    In another preferred embodiment of this aspect of the invention, the host of step (b) are
    Expression bacteria More preferably the expression bacteria are BL21 (DE3). The 35 expression bacteria BL21 (DE2) are chemically Escherichia coli cells
    competent, which has a suitable genotype for protein transformation and expression, and whose genome is known (Genome sequences of Escherichia coli B strains REL606 and BL21 (DE3). Jeong H, et al. J. Mol. Biol. 2009 Dec eleven).
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    A competent bacterium is characterized by a weakened bacterial wall and by
    5 it is much easier to capture a foreign DNA through a process of thermal or electrical shock (transformation). Expression bacteria are used for protein production. Here, the expression bacteria are those that possess the necessary machinery to overexpress the inserted cDNA and produce the recombinant protein.
    In another preferred embodiment of this aspect of the invention, the integration of the nucleotide sequence SEQ ID NO: 1 of step (a) is performed by a ligation process.
    For the completion of the ligation process, the insert mixture: plasmid was resuspended
    15 in the product In-Fusion Dry-Down pellet (Clontech). In-Fusion Dry-Down pellet is a lyophilisate that contains the In-Fusion enzyme, which favors the attachment of the insert to the plasmid thanks to homology in the nucleotide sequence present in both.
    Therefore, in another preferred embodiment of the invention, lyophilisate is used in the ligation
    20 comprising the enzyme In-Fusion. This is a Poxvirus DNA polymerase with 3'-5 'exonuclease activity, which is capable of binding single-stranded DNA molecules that have short and homologous sequences at their ends, such as an amplified PCR product and a vector.
    In another preferred embodiment, the insert was synthesized using nucleotide sequence primers SEQ ID NO: 3 and SEQ ID NO: 4.
    Another aspect relates to the use of a protein comprising the amino acid sequence SEQ ID NO: 2, or to the recombinant protein of the invention (soluble IFNAR2, sIFNAR2 or IFNAR2.3), obtained by other non-recombinant means, for use in the prevention, control, treatment and / or relief of a viral disease; or, alternatively, to the use of a protein comprising the amino acid sequence SEQ ID NO: 2 or to the recombinant protein of the invention, or the protein of the invention (soluble IFNAR2, sIFNAR2 or IFNAR2.3) obtained by other non-recombinant means, in the preparation of a medicine for the prevention, control, treatment and / or relief of a viral disease. It is preferable that the protein of the invention (soluble IFNAR2, sIFNAR2 or IFNAR2.3) is
    image9
    recombinant, and even more preferably obtained by the method described in the present invention, since the methods of obtaining and purifying described are advantageous, can be obtained by any method known in the state of the art for obtaining proteins.
    5
    The term "viral disease" refers to the clinical manifestation of a viral infection. A viral infection refers to the contamination, immune response and structural damage of a host, caused by a virus, that is, that there is invasion with cell or tissue injury caused by said viruses or their products. The infection can be local or systemic.
    10 The term "virus" refers to an acellular microscopic infectious agent that only has the ability to multiply within the cells of other organisms.
    Viruses are classified into different groups:
    15  Group I: double stranded DNA virus.  Group II: single stranded DNA virus.  Group III: double stranded RNA virus.  Group IV: positive single stranded RNA virus.  Group V: negative single stranded RNA virus.
    20  Group VI: retrotranscribed single stranded RNA virus.  Group VII: retrotranscribed double stranded DNA virus.
    Viral diseases that can be prevented, controlled, treated and / or relieved with the recombinant protein of the invention are preferably selected, although without
    25 Limit ourselves, from the list comprising: hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), hepatitis F or G virus (HBV), human immunodeficiency virus (HIV), respiratory syncytial virus, influenza virus, encephalomyocarditis virus, vesicular stomatitis virus or any combination thereof.
    30 HAV is a Group IV virus that belongs to the family Picomaviridae and the genus Hepatovirus. HBV is a Group VII virus that belongs to the Hepadnaviridae family and the genus Orthohepadnavirus. HCV is a Group IV virus that belongs to the Flaviviridae family and the genus Hepacivirus. VHD is a Group V virus that belongs to the
    35 Deltaviridae family and the genus Deltavirus. HEV is a Group IV virus that belongs to the Hepeviridae family. VHF is a single stranded DNA virus. VHG is an RNA virus also known as "GB virus C" or "GBVC".
    image10
    HIV is Group VI virus that belongs to the Retroviridae family and the genus Lentivirus. 5 There are two species of this virus: HIV Type 1 and HIV Type 2.
    Respiratory syncytial virus (RSV) is a single-stranded RNA virus in the negative direction of the Paramyxovirus (Paramyxoviridae) family, which includes common respiratory viruses such as those that cause measles and mumps. The VSR is a member of the subfamily of
    10 Pneumovirus.
    The influenza virus is a Group V virus of the Orthomyxoviridae family. There are different genera of viruses that produce influenza: InfluenzavirusA, InfluenzavirusB and InfluenzavirusC.
    15 Encephalomyocarditis virus is an RNA virus belonging to the genus Cardiovirus of the family. Enteroviruses, rhinoviruses, aftoviruses and hepatoviruses belong to the same family.
    The vesicular stomatitis virus is a Group V virus belonging to the family
    20 Rhabdoviridae and the genus Vesiculovirus. There are two serotypes of the vesicular stomatitis virus that are divided into different subtypes.
    USES OF THE ANTIBODIES AND COMPOSITIONS OF THE INVENTION.
    As demonstrated in the examples of the invention, the recombinant protein of the invention, and / or the sIFNAR2 protein, can be used for the prevention, control, treatment and / or relief of a viral disease. In addition, the use of the antibodies or fragments thereof capable of binding to the recombinant protein of the invention are
    30 also an object of the present invention. These antibodies or fragments thereof can be easily obtained from antisera.
    The antisera for the recombinant protein described in the present invention can be generated by standard techniques, for example, by injection of the recombinant protein of the invention into an appropriate animal and collection and purification of the animal antisera. Antibodies or fragments thereof that bind SEQ ID NO: 2, or
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    A variant sequence thereof according to the invention can be identified by standard immunoassays. The antibodies thus obtained (hereinafter, antibodies of the invention) can be used for the diagnostic method of the invention. Preferably, the antibodies or fragments thereof are monoclonal antibodies.
    Thus, in another aspect the invention relates to the use of an antibody or a fragment thereof that specifically recognizes the recombinant protein of the invention, hereinafter antibody of the invention, for use as a medicament for prevention, control, treatment and / or relief of a viral disease; or, alternatively, to the use of an antibody or a fragment thereof that specifically recognizes the recombinant protein of the invention, in the preparation of a medicament for the prevention, control, treatment and / or relief of a viral disease. Antibodies contemplated in the context of the present invention include polyclonal antisera, purified IgG molecules, supernatants or ascites containing monoclonal antibodies, Fv, Fab, Fab 'and F (ab') 2 fragments, ScFvdiabodies, triabodies, tetrabodies and humanized antibodies .
    Another aspect of the invention relates to the use of a composition, hereafter
    Composition of the invention, comprising:
    a) a protein comprising the amino acid sequence SEQ ID NO: 2,
    b) the recombinant protein of the invention,
    c) the protein of the invention (soluble IFNAR2, sIFNAR2 or IFNAR2.3) obtained by
    other non-recombinant media, and / or
    d) the antibody, or a fragment thereof, of the invention, in the preparation of a medicament for the prevention, control, treatment and / or relief of a viral disease.
    Said composition may be a pharmaceutical composition. Therefore, another aspect of the invention relates to pharmaceutical compositions, hereinafter pharmaceutical compositions of the invention, comprising at least one of the polynucleotides of the invention, polypeptides of the invention or its mature form, an antibody of the invention. , or a fragment thereof, the recombinant protein of the invention, the protein of the invention (soluble IFNAR2, sIFNAR2 or IFNAR2.3) obtained by other non-recombinant means, and / or accompanied by a pharmaceutically acceptable excipient. For use in medicine, the compounds and combinations of compounds of the invention can be formulated together with an excipient that is pharmaceutically acceptable. Preferred excipients for use in the present invention include sugars, starches, celluloses, gums, proteins and others. In a particular embodiment, the pharmaceutical composition of the invention will be formulated in a solid dosage pharmaceutical form (e.g., tablets, capsules, dragees, granules, suppositories, etc.) or
    image11
    5 liquid (eg, solutions, suspensions, emulsions, etc.), but not limited. In another particular embodiment, the pharmaceutical compositions of the invention can be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intrarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteric, topical, sublingual or rectal.
    10 "Adjuvants" and "pharmaceutically acceptable carriers" refer to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and include, but are not limited to, solids, liquids, solvents or surfactants Pharmaceutically acceptable vehicles
    15 that can be used in the present invention are the vehicles known in the state of the art.
    The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as age, weight, sex or tolerance, of the individual at
    20 to be administered. In the sense used in this description, the term "therapeutically effective amount" refers to the amount of the pharmaceutical composition of the invention that produces the desired effect and, in general, will be determined, among other causes, by the characteristics of such compounds and of said pharmaceutical composition and the therapeutic effect to be achieved.
    In a preferred embodiment of this aspect of the invention, the composition of the invention comprises another active ingredient. Preferably, the active substance is selected from the list consisting of other antivirals, analgesics, antipyretics, decongestants or other active ingredients used in the treatment of viral diseases.
    In another preferred embodiment, the composition of the invention further comprises a pharmaceutically acceptable carrier. In another preferred embodiment, the composition of the invention further comprises another active ingredient or therapeutic agent. Said therapeutic agent is preferably selected from an analgesic agent (in the treatment of the
    35 inflammation and pain) or an anti-infective agent (in the prevention of infection).
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    In particular, non-limiting examples of therapeutic agents useful according to the invention include the following therapeutic categories: analgesics, such as non-steroidal anti-inflammatory drugs, opioid agonists and salicylates; anti-infective agents, such as anthelmintics, antianaerobics, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporins, macrolide antibiotics, various beta-lactam antibiotics, penicillins, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, antimycobacterial antibiotics, antimycobacterial antibiotics antimalarial agents, antiviral agents, antiretroviral agents, scabicides, anti-inflammatory agents, anti-inflammatory corticosteroids, local anti-inflammatory drugs / topical anesthetics, topical anti-infective antifungal agents, topical anti-infective antiviral agents, electrolytic agents such as alkalinizing agents, alkalinizing agents of carbonic anhydrase, diuretics, loop diuretics, osmotic diuretics, potassium-sparing diuretics, thiazide diuretics, electrolyte supplements , and uricosuric agents, enzymes, such as pancreatic enzymes and thrombolytic enzymes, gastrointestinal agents, such as antidiarrheals, antiemetics, gastrointestinal gastrointestinal anti-inflammatory agents, salicylate anti-inflammatory agents, gastric ulcer antacids, inhibitory agents antiulcerous acid pump agents, gastric mucosa antiulcer agents H2 blockers, antiulcer agents, digestant choletholytic agents, emetics, laxatives and stool softeners and prokinetic agents, general anesthetics such as inhaled halogenated anesthetics, inhalation anesthetics, intravenous anesthetics, barbiturates, intravenous anesthetics Intravenous route of opioids and intravenous anesthetic agonists, hormones and hormonal modifiers, such as abortive, adrenal corticosteroid agents, adrenal agents, androgens, antiandrogens, immunobiological agents, such as immunoglobulins, immunosuppressants, toxoids, and vaccines; local anesthetics, such as amide from local anesthetics and local ester-type anesthetics, musculoskeletal agents, such as anti-gout anti-inflammatory agents, anti-inflammatory corticosteroids, agents, gold compounds anti-inflammatory immunosuppressive agents, anti-inflammatory agents, drugs Nonsteroidal anti-inflammatory drugs (NSAIDs), salicylate anti-inflammatory agents, minerals and vitamins, such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and vitamin K.
    In a particular embodiment, therapeutic agents useful according to the above categories include: (1) analgesics in general, such as lidocaine or its derivatives, and non-steroidal anti-inflammatory drugs (NSAIDs) analgesics, including diclofenac, ibuprofen,
    image12
    ketoprofen, naproxen and, (2) agonist opioid analgesics, such as codeine, fentanyl, hydromorphone and morphine, (3) salicylate analgesics, such as aspirin (ASA), (4) H1 antihistamine blockers, such as terfenadine, clemastine and (5) anti-infective agents, such as mupirocin; (6) anti-infective anti-anaerobic agents, such as chloramphenicol and clindamycin; (7) antifungal anti-infective antibiotics, such as amphotericin B, clotrimazole, fluconazole and ketoconazole; (8) against the infectious macrolide antibiotic, such as azithromycin and erythromycin; (9) various anti-infective beta-lactams, such as aztreonam and imipenem; (10) anti-infective penicillin antibiotic, such as nafcillin, oxacillin, penicillin G, penicillin V and; (11) quinolone 10 anti-infective antibiotics, such as ciprofloxacin and norfloxacin; (12) anti-infective tetracycline antibiotic, such as doxycycline, minocycline and tetracycline; (13) anti-infective antimicobacterial antituberculosis agents, such as isoniazid (INH), rifampicin and; (14) anti-infective antiprotozoa, such as atovaquone and dapsone; (15) anti-infective antiprotozoal antimalarials, such as chloroquine and pyrimethamine; (16) 15 anti-infective anti-retrovirals, such as ritonavir and zidovudine; (17) against the infectious antiviral agents, such as acyclovir, ganciclovir, interferon alfa, and rimantadine; (18) topical anti-infective antifungals, such as amphotericin B, clotrimazole, miconazole, nystatin and; (19) topical anti-infective antivirals, such as acyclovir; (20) electrolytic and renal agents, such as lactulose; (21) loop diuretics, such as furosemide, (22) 20 potassium-sparing diuretics, such as triamterene; (23) thiazide diuretics, such as hydrochlorothiazide (HCTZ), (24) uricosuric agents, such as probenecid; (25) enzymes, such as RNase and DNase; (26) antiemetics, such as prochlorperazine; (27) gastrointestinal inflammatory salicylate anti-agents, such as sulfasalazine; (28) gastric acid pump anti-inhibitor ulcer agents, such as omeprazole; (29) 25 H2 blockers, anti-ulcer agents, such as cimetidine, famotidine, nizatidine and ranitidine; (30) digestives, such as pancrelipase; (31) prokinetic agents, such as erythromycin (32; ester) local anesthetics, such as benzocaine and procaine; (33) anti-inflammatory corticosteroid musculoskeletal agents, such as beclomethasone, betamethasone, cortisone, dexamethasone, hydrocortisone, and prednisone; (34) 30 immunosuppressive anti-inflammatory musculoskeletals, such as azathioprine, cyclophosphamide and methotrexate; (35) non-steroidal anti-inflammatory musculoskeletal (NSAIDs), such as diclofenac, ibuprofen, ketoprofen, ketorlac, and naproxen; (36) minerals, such as iron, calcium and magnesium; (37) Vitamin B compounds, such as cyanocobalamin (vitamin B12) and niacin (vitamin B3); (38) vitamin C compounds, such as ascorbic acid, and (39) vitamin D compounds, such as
    calcitriol
    image13
    As used herein, the term "active substance", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity
    5 or another different effect on the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.
    In another preferred embodiment of this aspect of the invention, the viral disease is generated by a virus that is selected from the list that consists of: hepatitis A virus (HAV), hepatitis B virus (HBV), virus hepatitis C (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), hepatitis F or G virus (HBV), virus
    15 human immunodeficiency (HIV), respiratory syncytial virus, influenza virus, encephalomyocarditis virus, vesicular stomatitis virus or any combination thereof.
    The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of
    20 diseases or prevention of unwanted physiological conditions in man and animals.
    The term "individual", as used in the description, refers to animals, preferably mammals, and more preferably, humans. The term "individual" does not
    25 intends to be limiting in any aspect, being able to be of any age, sex and physical condition.
    Type I interferons (alpha, beta and omega) exert their action through interaction with the IFNAR membrane receptor, formed by two IFNAR1 and IFNAR2 subunits. The
    The IFNAR2 subunit of the receptor undergoes alternative mRNA processing that results in three different forms: a short form (IFNAR2b), a functionally active long form (IFNAR2c) and the soluble form (sIFNAR2, IFNAR2.3 or IFNAR2a). Only IFNAR2c acts as a functional receptor together with IFNAR1 and is able to mediate the biological effects of IFNβ, through the activation of the JAK-STAT signaling cascade.
    35
    image14
    Multiple variants of the transcript encoding at least two different isoforms have been found for this gene. The amino acid sequence of sIFNAR2 is found with access number in GenBank (NCBI) L41943.1 and in SEQ ID NO: 2. Said SEQ ID NO: 2 is represented by the following amino acid sequence:
    5
    MLLSQNAFIFRSLNLVLMVYISLVFGISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTH YTLLYTIMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGNTTLFSCSHNF WLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKGNMS GNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESEFS.
    In the context of the present invention, sIFNAR2 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 2, and which would comprise various variants from: a) molecules of nucleic acid encoding a polypeptide comprising the sequence
    Amino acid of SEQ ID NO: 2, b) nucleic acid molecules whose complementary strand hybrid with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the code genetic,
    D) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 60%, 70%, 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 2, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the IFNAR2.3 protein. Among these nucleic acid molecules is the collection in the sequence of the
    25 GenBank (NCBI) L41943.1 and SEQ ID NO: 1. Said SEQ ID NO: 1 is represented by the following nucleotide sequence:
    agatgtaaaagtcaagagaagactctaaaaatagcaaagatgcttttgagccagaatgccttcatcttcagatcacttaatttggttc tcatggtgtatatcagcctcgtgtttggtatttcatatgattcgcctgattacacagatgaatcttgcattttcaagatat
    30 tccggtccatcttatcatgggaattaaaaaaccactccattgtaccaactcactatacattgctgtatacaatcatgagtaaaccaga agatttgaaggtggttaagaactgtgcaaataccacaagatcattttgtgacctcacagatgagtggagaagcacacacgaggc ctatgtcaccgtcctagaaggattcagcgggaacacaacgttgttcagttgctcacacaatttctggctggccatagacatgtcttttg aaccaccagagtttgagattgttggttttaccaaccacattaatgtgatggtgaaatttccatctattgttgaggaagaattacagtttg atttatctctcgtcattgaagaacagtcagagggaattgttaagaagcataaacccgaaataaaaggaaacatgagtggaaattt
    35 cacctatatcattgacaagttaattccaaacacgaactactgtgtatctgtttatttagagcacagtgatgagcaagcagtaataaag tctcccttaaaatgcaccctccttccacctggccaggaatcagaattttcataactttttagcctggccatttcctaacctgccaccg
    5
    10
    fifteen
    twenty
    25
    30
    35

    gaagccatggatatggtggaggtcatttacatcaacagaaagaagaaagtgtgggattataattatgatgatgaaagtgatagcg atactgaggcagcgcccaggacaagtggcggtggctataccatgcatggactgactgtcaggcctctgggtcaggcctctgcca cctctacagaatcccagttgatagacccggagtccgaggaggagcctgacctgcctgaggttgatgtggagctccccacgatgc caaaggacagccctcagcagttggaactcttgagtgggccctgtgagaggagaaagagtccactccaggacccttttcccgaa gaggactacagctccacggaggggtctgggggcagaattaccttcaatgtggacttaaactctgtgtttttgagagttcttgatgacg aggacagtgacgacttagaagcccctctgatgctatcgtctcatctggaagagatggttgacccagaggatcctgataatgtgcaa tcaaaccatttgctggccagcggggaagggacacagccaacctttcccagcccctcttcagagggcctgtggtccgaagatgct ccatctgatcaaagtgacacttctgagtcagatgttgaccttggggatggttatataatgagatgactccaaaactattgaatgaactt ggacagacaagcacctacagggttctttgtctctgcatcctaacttgctgccttatcgtctgcaagtgttctccaagggaaggagga ggaaactgtggtgttcctttcttccaggtgacatcacctatgcacattcccagtatggggaccatagtatcattcagtgcattgtttacat attcaaagtggtgcactttgaaggaagcacatgtgcacctttcctttacactaatgcacttaggatgtttctgcatcatgtctaccaggg agcagggttccccacagtttcagaggtggtccaggacc ctatgatatttctcttctttcgttcttttttttttttttttgagacagagtctcgttct gtcgcccaagctggagcgcaatggtgtgatcttggctcactgcaacatccgcctcccgggttcaggtgattctcctgcctcagcctc cctcgcaagtagctgggattacaggcgcctgccaccatgcctagcaaatttttgtatttttagtggagacaggattttaccatgttggc caggctggtctcgaactcctgacctcaagtgatctgccctcctcagcctcgtaaagtgctgggattacaggggtgagccgctgtgc ctggctggccctgtgatatttctgtgaaataaattgggccagggtgggagcagggaaagaaaaggaaaatagtagcaagagct gcaaagcaggcaggaagggaggaggagagccaggtgagcagtggagagaaggggggccctgcacaaggaaacaggg aagagccatcgaagtttcagtcggtgagccttgggcacctcacccatgtcacatcctgtctcctgcaattggaattccaccttgtcca gggataaaacctgaatgccatattttaagttaaaaaaaaaaaa gccctccccagttaaagtggggaagacagactttaggatcacgtgtgtgactaatacagaaaggaaacatggcgtcggggaga.
    The terms "polynucleotide" and "nucleic acid" are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA).
    The terms "amino acid sequence", "peptide", "oligopeptide", "polypeptide" and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may be coding or non-coding, Chemically or biochemically modified.
    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

    EXAMPLES OF THE INVENTION 5 Example 1: Evaluation of the antiviral capacity of recombinant sIFNAR2 by the cytopathic effect test or Bioassay.
    It is the technique recommended by the World Health Organization (WHO) to evaluate antiviral capacity and for the detection of antibodies against IFNß. This technique is
    10 based on the antiviral capacity of IFNß, so that a cell culture (line A549), when infected with a specific virus (encephalomyocarditis virus), is protected by the presence of IFNß, unless neutralizing antibodies are present in the patient's serum that block the action of said IFNβ.
    15 We have carried out a series of bioassays in the presence of recombinant sIFNAR2 which has allowed us to verify that our recombinant sIFNAR2 has an important antiviral capacity while living cells remain, acting in a similar way to IFNβ.
    20 Figure 1 illustrates a representative bioassay performed in the presence of sIFNAR2. Greater intensity in the violet color corresponds to a greater cell survival after infection of the cell culture with the encephalomyocarditis virus (EMC). The different experimentation scenarios contemplated in Figure 1 are the following:
    25  Columns 1.2: IFNß Standard. It begins with a concentration of 20UI of IFNß and serial dilutions are made. As the concentration decreases, a degradation of color is observed because the cells are less protected by the action of IFNβ.  Columns 3.4: IFNß is added diluting from 20UI and a constant concentration
    30 of 30 ug / ml of sIFNAR2 throughout the entire column. The cells remain alive in all dilutions, with no degradation of color in any of the wells. As the IFNß stops protecting because it is diluting, sIFNAR2. Continue to protect the cells from the virus.  Columns 5-6 superior: 8 replicates adding sIFNAR2 60ug / ml. It can be seen
    35 that the cells remain alive, being protected from CV.
    image15
     Columns 5-6 lower: 8 replicates adding sIFNAR2 30ug / ml. It can be seen that the cells remain alive, being protected from viral control.
     Columns 7-8 upper: 8 replicates adding sIFNAR2 15 ug / ml. It can be seen that the cells remain alive, being protected from CV.
    5  Columns 9-10: 30ug / ml of sIFNAR2 was added to the entire column on the third day of the test, while the virus was added, thus observing a greater antiviral capacity.
     Upper columns 11-12: Cellular control (CC): only A549 cells;  Lower 11-12 columns: Viral Control (CV): A549 cells infected with the virus
    10 encephalomyocarditis, unprotected. Figure 4 consists of a Bioassay plate showing the antiviral capacity of sIFNAR2. It is shown that B18R, which is an inhibitor of IFNβ, blocks the action of IFNß but does not block the action of recombinant sIFNAR2 protein, it is observed how cells remain protected in the presence of B18R + sIFNAR2.
    15  Columns 1,2: IFNß Standard. It begins with a concentration of 20UI of IFNß and serial dilutions are made. As the concentration decreases, color degradation is observed because the cells are less protected by the action of IFNβ.  Columns 3.4: Combination of IFNß / sIFNAR2. IFNß is added diluting from
    20 20UI and sIFNAR2 diluting from a concentration of 60 ug / ml. It is observed how the presence of the two molecules slightly increases the protection with respect to each of them separately.
     Columns 5-6: sIFNAR2 60ug / ml is added in the first well and serial dilutions are made. It can be seen that the cells remain alive in the first 25 wells and the viability decreases as the concentration of
    sIFNAR2.
     Columns 7-8 upper: 4 replicates adding sIFNAR2 60 ug / ml and B18R (a potent IFNβ inhibitor) It can be seen that the cells remain alive, being protected from CV. The IFNβ inhibitor does not inhibit sIFNAR2.
    30  Columns 9-10 superior: 4 replicates adding IFNß 20U and B18R (a potent inhibitor of IFNß). It can be seen that the cells die since B18R inhibits IFNβ.
     Columns 7-8 lower: 4 replicas with IFN80 (added the same day as the virus). Protection is observed. 35  Columns 9-10 superior: 4 replicates with IFN40 (added the same day as the virus). Protection is observed.
    image16
     Columns 11-12 superior: Cellular control (CC): only A549 cells.  Lower 11-12 columns: Viral Control (CV): A549 cells infected with encephalomyocarditis virus, unprotected. 5 Example 2: Production of the SIFNAR2 recombinant protein
    Choice of cloning vector
    The prokaryotic expression system chosen is the pre-linearized vector pEcoli-Cterm 6xHN
    10 Linear (Clontech). The resulting protein will have a histidine-asparagine tail fused at the carboxy terminal end that will serve for purification.
    The structure of the vector where the insert was integrated with the nucleotide sequence of our protein of interest is detailed in the figures.
    15 The expression system of pEcoli Cterm 6xHN Linear is based on the T7 strong promoter expression system, controlled by the LacZ operon which in turn is inducible by IPTG (Isopropyl-β-D-thiogalactopyranoside). In addition, the plasmid has an ampicillin resistance gene that allows the selection of clones containing the plasmid.
    20 For the production of the protein, the machinery of the expression bacteria BL21 (DE3), which use the T7 promoter, was used. The BL21 bacteria (DE3) contain a chromosomal copy of the T7 RNA polymerase gene, which in turn is under the control of the IPU-inducible lacUV5 promoter.
    25
    DNA insert synthesis
    The first point in the design of the cloning strategy was the synthesis of the insert. For this, all the information about the SIFNAR2 sequence was collected, such as the signal sequence
    30 of the peptide, post-translational modifications, the biochemical characteristics of the protein, the domains thereof, etc. All this information was obtained from the UNIPROT database (http://www.uniprot.org/uniprot/P48551), which houses the amino acid sequences of the proteins and their biochemical characteristics.
    35 The SIFNAR2 mRNA sequence was obtained from the NCBI NUCLEOTIDE database (http://www.ncbi.nlm.nih.gov/nuccore.)
    image17
    Following the manufacturer's guidelines, the primers had to meet the following requirements: The 5´ end:
    5  Contain 15 homologous bases with the 15 bases at the end of the DNA fragment of the vector where it will be inserted.
    The 3´ end:  Have 15 homologous bases with the ends of the gene to be inserted.  A length between 18-25 bp and a GC content of 40-60%.
    10  Absence of the start (ATG) and stop codon in the sequence to be amplified.  Absence of the signal sequence.
    Taking into account these premises, the primers gave a product after the 638 bp amplification. The sequences of the primers were:
    fifteen
    Direction (sequence SEQ ID NO. 3): 5´TAAGGCCTCTGTCGACATTTCATATGATTCGCCTGATTACACGATG 3`
    Antisense (sequence SEQ ID NO. 4): 20 5´CAGAATTCGCAAGCTTTGAAAATTCTGATTCCTGGCCAGGTGGAA 3´
    The insert was synthesized by conventional PCR from the primers designed in the previous point, using a high fidelity Taq and using as a template a cDNA from a mixture of commercial human cDNA. The optimal conditions of
    25 concentrations, temperature and times for insert synthesis were as follows:
    Table 1. Summary of conventional PCR reagents for insert synthesis.
    Reagents Volume / SampleFinal concentration / sample
    Rnasas free water 40 μl
    Sense primer (20 μM) 1 μl0.4 μM
    Antisense primer (20 μM) 1 μl0.4 μM
    Dntp (10 μM) 1 μl0.2 μM
    5X buffer 5 μl1X
    Pfu High Fidelity 1 μl
    image18
    Temperature conditions:
    Table 2. Summary of temperature conditions for insert synthesis by conventional PCR.
    Stage TemperatureWeatherCycles
    Initial denaturation 95 ° C3 minone
    Denaturalization 95 ° C20 sec40 cycles
    Banding 60.4 ° C20 sec
    Extension 72 ° C30 sec
    Final extension 72 ° C10 minutesone
    Final stage 4ºCInfinite
    The final product obtained from the PCR was separated according to its size by means of the horizontal electrophoresis technique in a 2% agarose gel dissolved in TAE buffer, together with the Gold View Nucleic Acid Satin Intercalator (Sbs Genetech) at a dilution 1/20. He
    The gel was subjected to a constant current of 80 V and was viewed in an ultraviolet transilluminator, which allowed to locate the band of interest based on the number of base pairs. The band located at the height of 638 bp was trimmed from the agarose gel with the help of a scalpel.
    15 The sequence of the amplified insert contained in the agarose was purified with the commercial kit QIAquick Gel Extraction (QIAGEN) following the manufacturer's instructions. At the end of the process an eluate was obtained, which was quantified with a spectrophotometer (Nanodrop, Thermo) before being stored at -20 ° C.
    20 Ligation Process
    Following the work scheme, the next point in the cloning process was the ligation process, that is, "sticking" the plasmid, the nucleotide sequence of sIFNAR2, which will give rise to the recombinant protein.
    25 To determine the concentrations and volumes of the insert and the plasmid, the Clontech commercial house, on its website offers a computer tool (http://bioinfo.clontech.com/infusion) to calculate the optimal quantities of the vector and the insert, for the ligation process from the known variables length of the vector and the insert. For the completion of the ligation process, the insert mixture: plasmid was resuspended
    image19
    5 in the product In-Fusion Dry-Down pellet (Clontech). In-Fusion Dry-Down pellet is a lyophilisate that contains the In-Fusion enzyme, which favors the attachment of the insert to the plasmid thanks to homology in the nucleotide sequence present in both. The ligation reaction was carried out in a thermocycler, at 37 ° C for 15 minutes followed by 15 minutes at 50 ° C and subsequently transferred to ice. Finally, the ligation product
    10 was resuspended in 40 µl of TE buffer (Tris-HCl, EDTA) at pH 8.
    Transformation in replicative bacteria
    The competent bacteria used were MAX Efficiency DH5α ™ Competent Cells
    15 (Invitrogen) which were transformed with the plasmid, following the following protocol: As a positive control of the transformation technique, 5 µl of the plasmid pUC19 (positive control) was added in an aliquot of competent bacteria and this mixture was gently resuspended. In parallel, the bacteria were transformed with the product
    20 ligation. To do this, 2.5 µl was added to an aliquot of competent bacteria and mixed gently. Next, both aliquots of bacteria (control and problem) were incubated for 30 minutes on ice. After this time, the samples were subjected to a thermal shock at 42 ° C for 45 seconds. Quickly, the samples were transferred to ice for 2 minutes and then 900 µl of medium was added
    25 SOC (which favors the transformation process). In order for the plasmid to express ampicillin resistance, the samples were incubated at 37 ° C, with 225 rpm stirring for 1 hour. Finally, the transformed bacteria were seeded at different volumes in LB-Agar plates supplemented with 100 µg / ml ampicillin and incubated overnight at 37 ° C.
    30
    Plasmid DNA purification and verification of reading frame
    After one night in the incubator, the bacteria had formed CFU (colony forming units). To assess the characteristics of each UFC, these were
    35 independently isolated with the help of a seeding thread and seeded in tubes with 4 ml of LB Broth medium supplemented with 100 µg / ml of ampicillin. These suspensions were incubated at 37 ° C overnight with agitation of 220 rpm along with a negative control that was a LB Broth tube without bacteria. Subsequently, the plasmid contained in the bacteria was purified, following the indications of the Promega kit (PureYield ™ Plasmid Miniprep System) as explained below:
    image20
    5
    The bacteria culture was aliquoted in 1.5 ml tubes and centrifuged at 16000g for 30 seconds in a microcentrifuge. From the product obtained, the supernatant was discarded and the precipitate was resuspended in 600 μl of water, to which 100 μl of cell lysis buffer was added and mixed by inversion. To this mixture was added 350μl of neutralizing solution 10 and mixed again by inversion. It was then centrifuged at 16000g for 3 minutes. The supernatant obtained was transferred to one of the minicolumns provided by the kit that retains the DNA. It was centrifuged again at 16000g for 15 seconds. Then, 200μl of wash solution was added to the minicolumn and centrifuged again for 15 seconds. Subsequently, 400μl of wash solution 15 was added to the minicolumn and centrifuged for 30 seconds. Finally, to elute the DNA that had been retained in the membrane, the minicolumn was transferred to a clean 1.5 ml microcentrifuge tube, 30μl of sterile water was added to the center of the membrane and incubated for 1 minute at room temperature. Finally, to obtain the purified plasmid DNA, it was centrifuged at 16000g for 15 seconds. The DNA
    Plasmid was quantified by absorbance in the spectrophotometer (Nanodrop, Thermo) and was stored at -20 until the time of use.
    At this point we had different isolated and frozen CFUs, but it was unknown if the plasmid possessed the insert, its complete sequence, the orientation in the open reading frame etc., therefore it should be verified that the plasmid complied with all the requirements
    desired For this, two tests were carried out:  Conventional PCR using as plasmid DNA the plasmid DNA.
     DNA sequencing: The positive plasmids in the PCR were sequenced to obtain the nucleotide sequence that would allow us to evaluate the sequence of the insert and check its orientation.
    Sequence insertion encompassed upstream sequences that coincided with the T7 promoter and downstream with the terminal T7 sequence. The sequences obtained 35 were aligned in the 5'3 'direction with the NBCI reference sequence of GeneBank number: CAA61940.1 by means of the Multalin bioinformatics program. Then you
    image21
    It shows the results obtained after the alignment that assured us the integrity of the sequence and orientation in the correct reading frame:
    Transformation in BL21 expression bacteria (DE3)
    5
    Once the clone containing the plasmid was verified with the correct conditions, the plasmid was transformed into the BL21 expression bacteria (DE3) for the production of the sIFNAR2 recombinant protein, following the same protocol described above for transformation into replicative bacteria and detection of the plasmid.
    10
    Induction of sIFNAR2 recombinant protein expression
    Under normal conditions, in the BL21 bacteria (DE3) transformed with the plasmid, the recombinant protein is not being expressed because its expression is repressed by the Lac repressor (LacI) that is bound to the Lac operon. To allow its expression, it is
    It is necessary to add IPTG that acts as an inducer by sequestering the repressor and allowing the T7 RNA polymerase to bind to the T7 promoter and carry out the transcription process. To induce the expression of the sIFNAR2 recombinant protein, the following protocol was followed: On the day before induction of protein production, a preculture of the protein was prepared.
    20 as follows:  BL21 bacteria (DE3) with the plasmid were grown in 4ml of LB-Broth supplemented with ampicillin at a final concentration of 100μg / ml and incubated overnight at 37 ° C with a stirring of 220 rpm.  The next day, the induction of protein expression was performed. To do this, the
    The culture of the previous day was diluted 1/10 in a final volume of 50 ml of LB-Broth medium supplemented with ampicillin and incubated at 37 ° C with a stirring of 220 rpm until reaching an optical density (D.O.) of 0.80-1 nm. At this time, the IPTG inducer was added to a final concentration of 0.5mM (previously established) and the culture was incubated for 4 hours at 37 ° C with agitation of 220 rpm. From
    30 This time the transcription process for protein expression began. After 4 hours of induction (previously optimized) the culture was collected and centrifuged at 1600g at 4 ° C for 20 minutes. The supernatant was discarded and the pellet stored at -80 ° C until later use.
    35 Recombinant Protein Extraction
    image22
    The expressed recombinant protein was located inside the bacteria. To access it and be able to purify it, the bacterial wall had to be broken by physical and chemical processes that are detailed below:
    5 The precipitate of bacteria stored at -80 ° C was thawed at room temperature. Next, 0.5 ml of bacterial lysis buffer was added for each milliliter of initial culture and resuspended with the help of a pipette. The resulting suspension was incubated for 1 hour at room temperature in rotation. After this time the sample was subjected to ultrasound in cycles of 5 pulses of 30 seconds on ice, and with
    10 an intensity of 40%. It was then ultracentrifigated at 15,000 g for 20 minutes at 4 ° C and with this the membranes of the proteins released from the bacteria were separated. After ultracentrifugation the supernatant was collected and passed through a 0.45 μm filter.
    Purification of the sIFNAR2 recombinant protein
    15 The product obtained after extraction contained the recombinant protein together with other bacterial proteins. To purify and isolate the sIFNAR2 recombinant protein, the affinity chromatography technique was used, so that the sIFNAR2 recombinant protein is retained by the histidine-asparagine tail it contains. The chosen columns are
    20 present in a volume of 1 ml and are filled with sepharose resin that have nickel ions attached. Nickel ions confer the ability to retain histidine-rich proteins and therefore the sIFNAR2 recombinant protein will be retained, among others. The protein release from the resin is produced by the addition of an imidazole-rich buffer that competes with the nickel binding site. The protocol is detailed below.
    25 followed:
    Before starting the purification process with affinity chromatography, the resin was washed and equilibrated with 10 ml of equilibration buffer. Then, the protein extract that contains our protein of interest, was contacted with the resin in rotation at
    30 4 ° C for 1 hour and subsequently, the resin was packed in the column. To remove the proteins not bound to the resin, it was washed with 10 ml of equilibration buffer. Finally, the proteins retained by nickel were eluted with 5 ml of elution buffer rich in imidazole and collected in 1 ml aliquots.
    35 Recombinant protein detection: Electrophoresis and Western blot
    image23
    The first step for protein detection was the electrophoresis in polyacrylamide gels and subsequently the transfer of proteins to a membrane. The protocol followed was: The samples were resuspended in 5x loading buffer and boiled at 100 ° C for 3
    5 minutes in a thermoblock. These were then loaded into a 12% polyacrylamide gel, immersed in an electrophoresis buffer and subjected to a constant current of 130 V. Once the electrophoresis was finished, the gel obtained was immersed in transfer buffer for a few minutes. The transfer was made in a semi-dry system in graphite sheets that previously
    10 had been moistened with water. The nitrocellulose membrane with a pore size of 0.45 µm was then activated by immersing it in water and subsequently equilibrated in transfer buffer. Subsequently, the sandwich was assembled; on the graphite plate; 9 transfer papers previously moistened in transfer buffer were placed, then the membrane on top of it and on it
    15 the gel that was to be transferred. To finish the sandwich, 9 dampened transfer papers were put back in transfer buffer. The transfer was carried out for 45 minutes with an intensity of 0.8 mA / cm2. Once the transfer was finished, the membrane was separated and blocked with blocking buffer for 2h at room temperature and with stirring. The block is a stage that
    20 prevents non-specific binding of antibodies to free membrane sites, these being blocked with milk casein. After blocking, the membrane was contacted with the primary anti-IFNAR2 Human antibody produced in rabbit (Abnova) 1/5000, previously established dilution, in blocking solution overnight at 4 ° C in rotation. The next day, the membrane was removed from the solution with antibody and
    25 washed with wash buffer. The membrane was incubated for an hour and a half with the anti-rabbit IgG antibody (Sigma-Aldrich) labeled with alkaline phosphatase, at a dilution 1/10000 in blocking solution. It was washed as in the previous point. To see the result of the western blot, the membrane was revealed by contacting it with a mixture formed by 200 µl of NBT / BCIP + 10ml of developing solution at room temperature
    30 until the appearance of a colored product. Finally, the reaction was stopped by discarding the developing solution and immersing it in stop solution, rich in magnesium ions that block the development of the colorimetric reaction by removing the NBT / BCIP.
    Recombinant protein analysis
    35
    image24
    After purification of the sIFNAR2 recombinant protein, it was analyzed. To do this, the proteins / bands of an SDS / PAGE acrylamide gel were cleaved and fragmented to perform the subsequent analysis of the peptide fingerprint by MALDI-TOF / TOF mass spectrometry.
    权利要求:
    Claims (7)
    [1]
    image 1
    1.-Use of the sIFNAR2 protein in the preparation of a medicine for the prevention, improvement, treatment and / or relief of a viral disease.
    5
    [2]
    2. The use of the sIFNAR2 protein according to the preceding claim, wherein the amino acid sequence of the sIFNAR2 protein is SEQ ID NO. 2, or an amino acid sequence with an identity of at least 80% with SEQ ID NO. 2, and in which the protein encoded by said amino acid sequence has the activity and characteristics
    10 structural sIFNAR2 protein.
    [3]
    3. The use of the sIFNAR2 protein according to any of claims 1-2, wherein the sIFNAR2 protein is a recombinant protein.
    4. The use of the sIFNAR2 protein according to any of claims 1-3, wherein the sIFNAR2 protein is obtained by non-recombinant means.
    [5]
    5. Use of a composition comprising the sIFNAR2 protein as defined by this protein in any of claims 1-4, in the preparation of a medicament
    20 for the prevention, improvement, treatment and / or relief of a viral disease, wherein said composition is a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier and / or pharmaceutically acceptable excipients.
    [6]
    6. The use of the composition according to the preceding claim, which further comprises another active ingredient.
    [7]
    7. The use of the composition according to the preceding claim, wherein the active ingredient is selected from the list consisting of other antivirals, analgesics, antipyretics, decongestants or other active ingredients used in the treatment of diseases
    30 viral
    [8]
    8. The use of a composition according to any of claims 5-7, wherein the viral disease is generated by a virus that is selected from the list that consists of: hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HBV), hepatitis F or G virus (HBV), virus
    30
    image2
    human immunodeficiency (HIV), respiratory syncytial virus, influenza virus, encephalomyocarditis virus, vesicular stomatitis virus or any combination thereof.
    31
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    ES2626002B1|2018-05-10|
    EP3424520A4|2019-10-09|
    EP3424520A1|2019-01-09|
    WO2017109257A1|2017-06-29|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2470816B1|2012-11-22|2015-04-01|Servicio Andaluz De Salud|Recombinant protein and uses in the diagnosis of multiple sclerosis|
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    PCT/ES2016/070917| WO2017109257A1|2015-12-21|2016-12-21|Method for obtaining ifnar recombinant protein, and use thereof as an antiviral|
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