• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Composition useful for producing vaccines containing particles having immunogenic properties characteristic of antigen HBsAg, said particles being more particularly characterized in that they also contain a receptor for polymerized human albumin. They are obtained by transformation of human or animal cells by a vector containing a DNA sequence coding for the S and pre-S regions of a gene of viral hepatitis B, said DNA sequence being placed under direct control of a promoter enabling the efficient transcription of said sequence in human or animal cells transformable by said vector.
    公开号:SU1625332A3
    申请号:SU854011814
    申请日:1985-11-06
    公开日:1991-01-30
    发明作者:Собзак Элиан；Мальпьес Ив；Мишель Мари-Луиз；Тиоллэ Пьер；Э.Штреек Рольф
    申请人:Энститю Пастер (Фирма)；Энститю Насьональ Де Ля Санте Э Де Ля Решерш Медикаль (Фирма)；Сантр Насьональ Де Ля Решерш Сьянтифик (Фирма)；
    IPC主号:
    专利说明:

    This invention relates to the field of medicine and can be used to produce hepatitis B vaccines.
    The composition used for the preparation of vaccines according to the invention contains almost spherical polypeptide particles (or is formed from these particles) that have immunogenic and immunological properties characteristic of the HBS Ag antigen and are 18-25 nm in size, especially 20-22 mm , and densities allowing their isolation in the density range of 1.20-1.22 g / mm in the CsCl-based density gradient, and such a degree of total purity that there are no Dane s particles (and HBS antigens, including NAF. The proposed composition differs that specified spherical particles also contain a polymerized human albumin receptor. In particular, the polypeptide particles of the invention may contain significant proportions of h polypeptides having a molecular weight of about 34,000 daltons, and such polypeptides are more than 10% , preferably more than 20% of the total number of polypeptides of these particles.
    Example 1. Construction of vectors.
    A. Construction of the pSVS plamid,
    C & IsD
    SP
    WITH
    with
    1C

    cm
    Plasmid pSVS allows expression of the S region (pre-S region and the S gene) under the control of the early promoter of the SV40 virus.
    A 2.3 kb Bglll fragment was isolated from pSRY. The pSRYu plasmid contains a two-link polymer (dimer) from the head to the tail of the HBV genome. This fragment starts at ten nucleotides before the ATC of the pre-S region and ends 1.1 kV after the stop codon of the TAA gene S. It contains the putative polyadenylation point of the informational ribonuclease (mRNA) receptor, and the transcription point of the gene S.
    The SV40 early promoter contained in the plasmid PSV 2 gpt (ATCC 37145), in which the gpt gene is used
    E. coli is fused with a PVuII Hindlll fragment of size 348 bp. SV40 early region. This fragment includes early and late promoters of a different origin than SV 40 replication, the point of initiation of transcription of early messengers, and 72 main pairs, which are repeated in sequence.
    The 2.3 kbp Bglll fragment from pCRY inserted the plasmid pSV into a single Hindlll point by linking the ends of the DNA fragment, which have a tendency to be revealed by Klenow polymerase DNA. After studying the recombinant plasmids, a clone (pSV H44) is selected in which the HBS coding region is oriented relative to the SV 40 promoter in such a way that it is expressed. Such a construction is confirmed by restrictive maps. The nucleotide sequence was determined sequentially at the junction of two SV 40 and HPV fragments.
    The pSVS plasmid is constructed from three fragments obtained from three plasmids: the 2.5 kv PVuI-XLol fragment obtained from pSVH4; a fragment of 1.9 kV Xhol BglHI, obtained from rSRY; fragment 1.0 -BamHI, PVuI from pBR 322
    The resulting plasmid
    pSVS (5.4 kV) differs from pSV H4 in the absence of the gpt region and from the SV40 DNA sequences following this sequence in the presence of the EcoRI fragment - BamHI of the plasmid pBR 322.
    B „Construction of the pSVS plamid.
    The fragment containing dhfr.

    0
    five
    $
    five
    five
    0
    0
    The pHTV dhfr plasmid, after linear alignment with the PVuI enzyme, was partially digested with the Hindlll enzyme, releasing some fragments, of which one fragment was 4400 Lp. (base pairs) contains LTR from MMTV, with DNA from dhfr, intron from tAg from SV 40, polyadenylation point in early SV 40 genes, EcoRI-PVuI fragment of pBR 322 plasmid.
    HBV DNA fragment.
    Plasmid pSVS is fragmented by PVuI, then Hindlll, thus releasing fragment 4676 bp containing the PVuI-PVuII Fragment containing the replication source pBR 322; replication source and early promoter of the SV 40 virus; a fragment of Bglll size 2,3 T. p. from HBV, containing the regions encoding the pre-S and S gene, as well as the polyadenylation signal of this gene; BamHI fragment - Hindlll plasmid pBR 322.
    Ligation of data fragments,
    After purification, these two fragments are joined at the PVuI and Hindlll sites so that the resistance of ampicillin pBR 322 is restored.
    In the recombinant transcription zone vector, the S region and the dhfr gene are oriented in the same direction and are separated by about 300 bp. plasmids pBR 322.
    DNAs encoding dhfr are also obtained from pSV2 dhfr (ATCC 337146), pSV3 dhfr (ATCC 37147) or pSVS dhfr (ATCC 31148).
    Locating the dhfr gene in such a way that it is under the control of a weak promoter (LTR from MMTV) and the HBV gene in such a way that it is under the control of a strong promoter (early promoter SV 40), it increases the efficiency of gene expression.
    Example 2. Transfection of animal cells.
    Transfer and expression of the pSVS dhfr plasmid into CHOo Transform CHO dhfr cells with the pSVS dhfr plasmid, the product of these cells is tested by radioimmunoassay (RIA) in the surface layer of cell cultures. 60% of the dhfr clones are HBS Ag. The rate of synthesis of Ag HBS was relatively low between 1 and 20 mg / 106 cells for 24 hours.
    Increased HBS sequences.
    Cultivation of CHO HBS kg1 clones in the presence of methotrixate (MTX), a folic acid analogue in the dhfr inhibitor, causes an increase in the number of reproductions of the dhfr gene, which leads to an increase in the amount of the dhfr enzyme.
    MTX concentrations of 50, 100 or 140 nM in the first stage and 1.5.10 or 25 µmol in the second stage Approximately 150 MTX-resistant clones are selected for the production of HBS Ag, while the increase in the synthesis of HBS Ag significantly varies from one clone to another, both in the first and in the second stage.
    The number of copies of the HBV sequence per cell in different clones was determined by hybridizing the cellulose filter according to the method using cloned HBV - DNA labeled with g32p as a sample. For the same dose of MTX, this number varies considerably from one clone to another in the range from 100 to 500 .
    The HBV sequences are present in an integrated form, the reforetic profiles for different clones are generally comparable.
    The number of specific RNA HBV sequences is analyzed by molecular hybridization, it is proportional to the rate of HBS production of AЈ. Moreover, for the same dose of MTX (50 nm), the amount of specific RNA is constant in various analyzed clones. Two classes of RNA have been identified. One main 2.1 kb and another minor 2.5 gn. OO The study of these RNAs via the nuclease S1 map shows the presence of the main initiation region on the pre-S at position 3157, corresponding to 2.1 mA RNA. O., and another minor initiation in the SV 40 promoter, corresponding to a PH of 2.5 kb,
    PRI me R 3 Analysis of particles HBS Ag
    The analysis is carried out on particles obtained by 37B5. The surface layers corresponding to several collections in the stationary phase for 24 hours are combined. Ag HBS particles are purified by two consecutive
    five
    0
    five
    100 ° C
    ultracentrifugation in a density gradient of CsCl followed by high-speed ultracentrifugation in a sucrose gradient. Ag HBS particles have a density of 1.20. In an electron microscope, they are found to be spherical particles with an average diameter of 22 nm, morphologically similar to particles of human origin. Tubular particles do not detect.
    After dissociation of particles at
    for 5 min in the presence of DTT, the polypeptides are analyzed by polyacrylamide gel electrophoresis in the presence of SDS. The gel is stained with silver salts. Three bands corresponding to 22,300, 26,100 and 34,000 daltons are observed. The relative staining intensity of these three bands allows the proportions of the three proteins to be determined, being 54, 19 and 27%. After 35 S is labeled with methionine in vivo, the purified particles are immunoprecipitated with anti-HBS serum, and then the proteins are analyzed by electrophoresis. On the autoradiogram, the three described polypeptides are detected in the same proportions.
    The presence of a pHSA receptor on the surface of the particles is detected by radioimmunoassay in the solid phase. Receptors are found in the surface layer, as well as on the purified particles.
    PRI me R 4. Definition of immunization.
    After introducing the alumina hydrate particles into the preparation to a concentration of 0.1%, this preparation is used to immunize Balb / c mice. The immunological strength of cell particles 5 is identical to the immunological strength of the hepatitis B vaccine, manufactured under the brand name HBV ACB (and obtained from human serum donors with a content of natural HBS Ag). 0 50% effective dose is 0.04 µg for cell particles and 0.03 µg for HBVAC (trademark of the Pasteur Institute).
    Thus, the vector pSVS, after integration into cellular DNA, allows the synthesis and isolation of empty shells of HBV virus in the form of particles of 22 nm. Removal of most of the HBV genome, and in particular the gene, cod0
    five
    0
    five
    capsid protein, eliminates the production of completely infectious virus particles. This vector also carries the transcription link of the dhfr brown alga gene, which, after being introduced into dhfr, provides for their increase in the presence of MTX by increasing the gene.
    Synthesis of Ag HBS through CHO blocks is very stable even in the absence of MTX. And this stability is maintained for six months, which is approximately 300 generations. In some cases, there is still a slight spontaneous increase in HBS produced by Ag.
    In addition, the invention provides clones that form immunogenic particles with significant
    genetic stability, j
    The 22 mm HBS Ag particles synthesized by CHO cells have the same immunogenicity as HBV DIA, which is a preparation of particles of serum origin. Ag HBS production rates are comparable to production rates for industrial use. Presence of pHSA receptor on top of
    five
    0
    five
    0
    of these particles is an additional argument for using this system in obtaining hepatitis B vaccine.
    权利要求:
    Claims (1)
    [1]
    Invention Formula
    The method of obtaining a polypeptide with immunogenic properties of HBS Ag, involving the construction of recombinant plasmid DNA from the inserted part of the hepatitis E virus DNA under the control of a viral promoter, transformation of the obtained DNA of cultured animal cell strains, selection of producer strains, cultivation, isolation and purification of the target product, that a recombinant plasmid DNA shaker dhfr is designed with the insertion of a 23 kb Bglll fragment of the hepatitis B virus under the control of the early promoter of SV 40, trans the obtained DNA strain CHO dhfr is formed, and the producer strain is selected in the presence of MTX at a concentration of 50, 100, or 150 nmol, then at a concentration of 5.10 or 20 mmol and blot hybridization with HBK-DNA used as a DNA probe labeled 3ip.
    类似技术:
    公开号 | 公开日 | 专利标题
    JP2634371B2|1997-07-23|Vector expressing DNA encoding hepatitis B surface antigen in vertebrate cells
    US6306625B1|2001-10-23|Method for obtaining expression of mixed polypeptide particles in yeast
    EP1294893B1|2006-03-15|Modification of hepatitis b core antigen
    US4977092A|1990-12-11|Expression of exogenous polypeptides and polypeptide products including hepatitis B surface antigen in yeast cells
    Kann et al.1994|Effect of core protein phosphorylation by protein kinase C on encapsidation of RNA within core particles of hepatitis B virus
    CA2025598C|2001-02-27|Chimaeric hepadnavirus core antigen proteins
    EP0013828B1|1987-03-25|Recombinant DNA, hosts transformed with it and processes for the preparation of polypeptides
    CA2405438C|2011-07-12|Hepatitis b core antigen fusion proteins
    Kuhröber et al.1997|DNA vaccination with plasmids encoding the intracellular | or secreted | form of the core protein of hepatitis B virus primes T cell responses to two overlapping Kb-and Kd-restricted epitopes.
    Phogat et al.2008|Analysis of the human immunodeficiency virus type 1 gp41 membrane proximal external region arrayed on hepatitis B surface antigen particles
    EP0304578A1|1989-03-01|Peptide comprising hepatitis B surface antigen
    JPH10508468A|1998-08-25|Expression of peptides in plants as virus coat protein fusions
    EP0300213A1|1989-01-25|Hepatitis a viral peptide particle immunogens
    US5098704A|1992-03-24|Hepatitis surface antigen particle vaccine
    SU1625332A3|1991-01-30|Method of producing polypeptide with immunogenic properties of hbsag
    US5324513A|1994-06-28|Composition useful for the fabrication of vaccines
    JPH09248191A|1997-09-22|Fused protein and granule thereof, diagnostic reagent
    Yim et al.1996|Poliovirus recombinants expressing hepatitis B virus antigens elicited a humoral immune response in susceptible mice
    CA1341021C|2000-06-13|Hepatitis surface antigen particle vaccine
    EP0182442B1|1990-07-11|Recombinant dna molecules and their method of production
    US4741901A|1988-05-03|Preparation of polypeptides in vertebrate cell culture
    Bisht et al.2001|Expression and purification of dengue virus type 2 envelope protein as a fusion with hepatitis B surface antigen in Pichia pastoris
    EP0062574A2|1982-10-13|Virus protein synthesis
    Voronkova et al.2002|Chimeric bacteriophage fr virus-like particles harboring the immunodominant C-terminal region of hamster polyomavirus VP1 induce a strong VP1-specific antibody response in rabbits and mice
    EP1117803B1|2006-12-13|Method for the production of |peptides by using truncated variants of the sv40 large t antigen with an intact n terminus
    同族专利:
    公开号 | 公开日
    JPS61500971A|1986-05-15|
    AU598181B2|1990-06-21|
    DE3588117T2|1997-03-20|
    WO1985003876A1|1985-09-12|
    KR850700214A|1985-12-26|
    KR930003609B1|1993-05-08|
    FR2560890A1|1985-09-13|
    AT141168T|1996-08-15|
    EP0732104A1|1996-09-18|
    EP0156712B1|1996-08-14|
    DE3588117D1|1996-09-19|
    CA1262532A|1989-10-31|
    JPH08839B2|1996-01-10|
    EP0156712A1|1985-10-02|
    AU4061085A|1985-09-24|
    FR2560890B1|1987-10-16|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US4113712A|1976-03-08|1978-09-12|The Green Cross Corporation|HBsAG Particle composed of single polypeptide subunits and the preparation procedure|
    CA1100037A|1977-03-11|1981-04-28|Chung-Mei Ling|Hb.sub.c ag coated on solid phase|
    IN151589B|1978-12-22|1983-05-28|Biogen Nv|
    JPS5721246B2|1979-07-05|1982-05-06|
    EP0038765B1|1980-04-22|1987-09-02|Institut Pasteur|Vaccine against viral hepatitis b, method and transformed eucaryotic cells for the preparation of this vaccine|
    FR2480780B1|1980-04-22|1985-12-06|Pasteur Institut|PROCESS FOR THE TRANSFORMATION OF CELLS, ESPECIALLY EUKARYOTES, BY CIRCULAR DNA SUCH AS THAT OF HEPATITIS B VIRUS AND PREPARATIONS CONTAINING THE EXPRESSION PRODUCTS OF SAID DNA|
    ZW18282A1|1981-08-31|1983-03-23|Genentech Inc|Preparation of polypeptides in vertebrate cell culture|
    JPS6321476B2|1982-08-20|1988-05-07|Kagaku Gijutsucho Chokan Kanbo|
    US4847080A|1984-03-07|1989-07-11|New York Blood Center, Inc.|Pre-S gene coded peptide hepatitis B immunogens, vaccines, diagnostics, and synthetic lipide vesicle carriers|
    AU579148B2|1984-03-09|1988-11-17|Scripps Clinic And Research Foundation|Synthetic hepatitis b virus vaccine including both t cell and b cell determinants|
    NL8401066A|1984-04-04|1985-11-01|Stichting Centraal Lab|PROCESS FOR PREPARING PREPARATIONS INCREASING HUMORAL AND CELLULAR IMMUNE ACTIVITY|IL79740D0|1986-08-17|1986-11-30|Yeda Res & Dev|Hepatitis vaccine|
    FR2608052B1|1986-12-10|1990-04-13|Pasteur Vaccins|PROCESS FOR THE PREPARATION OF A HEPATITIS B VACCINE AND VACCINE OBTAINED|
    IL86833D0|1987-06-22|1988-11-30|Medico Labs|Peptide containing immunogenic particles|
    EP0304578B1|1987-06-22|2001-10-24|Medeva Holdings Bv|Peptide comprising hepatitis B surface antigen|
    AU619753B2|1987-06-22|1992-02-06|Medeva Holdings B.V.|Hepatitis b surface antigen vaccine|
    EP0328123A1|1988-02-09|1989-08-16|Eugene Tech International, Inc.|Heterogeneous, pre-S rich hepatitis B surface antigen|
    EP0491077A1|1990-12-19|1992-06-24|Medeva Holdings B.V.|A composition used as a therapeutic agent against chronic viral hepatic diseases|
    FR2711670B1|1993-10-22|1996-01-12|Pasteur Institut|Nucleotide vector, composition containing it and vaccine for immunization against hepatitis.|
    JP4085231B2|2000-02-28|2008-05-14|株式会社ビークル|Protein hollow nanoparticles, substance carrier using the same, and method for introducing substance into cells|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    FR8403564A|FR2560890B1|1984-03-07|1984-03-07|COMPOSITION USEFUL FOR THE MANUFACTURE OF VACCINES CONTAINING PARTICLES CARRYING THE SURFACE ANTIGEN OF HEPATITIS B VIRUS AND THE POLYMERIZED HUMAN SERUM ALBUMIN RECEPTOR, ANIMAL CELLS CAPABLE OF PRODUCING SUCH PARTICLES|
    [返回顶部]