WO2005116218A1 - Gene de fusion d'un antigene de surface de l'hepatite b avec une extremite carboxyle possedant un determinant immune pres2 et proteine de celui-ci - Google Patents

Gene de fusion d'un antigene de surface de l'hepatite b avec une extremite carboxyle possedant un determinant immune pres2 et proteine de celui-ci Download PDF

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Publication number
WO2005116218A1
WO2005116218A1 PCT/CN2005/000737 CN2005000737W WO2005116218A1 WO 2005116218 A1 WO2005116218 A1 WO 2005116218A1 CN 2005000737 W CN2005000737 W CN 2005000737W WO 2005116218 A1 WO2005116218 A1 WO 2005116218A1
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hepatitis
protein
antigen
recombinant
surface antigen
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PCT/CN2005/000737
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English (en)
Chinese (zh)
Inventor
Changyao Tan
Liming Jiang
Yonghong Ge
Jin Yuan
Ou Jin
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Chengdu Institute Of Biological Products
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Priority to CNB2005800006774A priority Critical patent/CN100413970C/zh
Publication of WO2005116218A1 publication Critical patent/WO2005116218A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a novel hepatitis B surface antigen coding gene, and in particular to a hepatitis B surface antigen fusion gene with a pre-S2 immunodeterminant at the carboxyl end constructed by a genetic engineering method.
  • the invention also relates to a recombinant vector of the gene and a protein encoded by the vector. Background technique
  • Hepatitis B is a serious and harmful infectious disease caused by the hepatitis B virus. It is widely distributed worldwide. It is estimated that there are about 120 million carriers of hepatitis B virus in China alone, of which about 10 million patients . At present, there is no effective treatment for hepatitis B, and hepatitis B vaccine immunization is a practical and effective way to prevent and control hepatitis B.
  • hepatitis B vaccines were made from hepatitis B surface antigen particles isolated from the plasma of hepatitis B virus carriers. Due to limited plasma sources and cost and safety considerations, these vaccines have been discontinued in most countries and regions. At present, widely used at home and abroad is a recombinant genetically engineered vaccine composed of hepatitis B virus surface antigen main protein (S) produced by recombinant yeast or CHO cells. Compared with blood-derived vaccines, genetically engineered vaccines have lower cost and better safety, and have achieved good immune effects.
  • S hepatitis B virus surface antigen main protein
  • this single S recombinant vaccine also has many shortcomings, such as too long immunization program, immune escape caused by HBV mutant strains and non-response or low response in some people. Therefore, it is still urgent to develop new vaccines with better immunogenicity and lower prices.
  • the hepatitis B virus envelope protein contains three components: large (L), medium (M), and main protein (S).
  • the S protein consists of 226 amino acids.
  • preS2 consisting of 55 amino acids ( preS2) region and a pre-SI (preSl) region composed of 119 amino acids (ad subtype), in which PreS2 and S—together constitute a middle protein, and preS1 and M—together constitute a large protein.
  • preS2 antigen is more immunogenic than S protein. Immunizing mice with hepatitis B surface antigen containing both preS2 and S regions can allow antibodies that do not respond to S antigen to produce antibody responses to DR., Thornton GB ., Neurath AR., Et al.
  • the synthetic preS2 polypeptide was used to confirm that its 26 amino acids at the amino terminus (Me-Gly 26 ) are the main antibody binding sites in this region.
  • the antibody of the synthetic polypeptide can neutralize the virulence of hepatitis B virus. (Neurath AR., Kent SBH., Parker K, et al. Antibodies to a synthetic peptide from the preS120-145 region of the hepatitis B virus envelope are virus neutralizing. Vaccine 1986, 4: 35-37).
  • the natural preS2 region contains multiple protease sensitive sites, especially the peptide bond between its 48th arginine and 49th threonine at the N-terminus is prone to cleavage by proteases, angky KE., Egan KM., Barendt JM., Et al. Characterization of purified hepatitis B surface antigen containing pre-S (2) epitopes expressed in saccharomyces cerevisiae. Gene 1988, 67: 229-245), causing the preS2 antigen to be easily degraded during the expression and preparation process and Inactivated.
  • preS2 and S genes contain their own translation initiation sites, when the structure of preS2 + S is expressed in animal cells, two products, medium protein and main protein, will be produced, thereby reducing the preS2 antigen in the product.
  • Content Hui J, Li Q Kong Y et al. Expression and Characterization of chimeric hepatitis B surface antigen particles carrying preS epitopes. Journal of Biotechnoly, 1999, 72: 49-59; Chinese patent 1067721C; Chinese patent CN 10585250.
  • S2 antigen is easily degraded and a single translation product can be obtained.
  • the present invention has a new design for the structure of the hepatitis B surface antigen containing the pre-S2 region.
  • An object of the present invention is to provide a new gene encoding a hepatitis B virus surface antigen, comprising a nucleotide sequence encoding a hepatitis B surface antigen S protein and a nucleotide sequence encoding a hepatitis B surface antigen pre S2 protein, which is characterized by The nucleotide sequence of amino acids 1-26 of the former S2 protein is linked to the 3 ′ end of the nucleotide sequence encoding the S protein.
  • a nucleotide sequence of SEQ ID NO. 1 is constructed. Shows the sequence of the new gene.
  • Yet another object of the present invention is to provide a protein having hepatitis B virus surface antigen S and pre-S2 antigenicity, the protein comprising an amino acid sequence encoded by the novel gene of the present invention.
  • the present invention has hepatitis B
  • the viral surface antigen S and pre-S2 antigenic protein are encoded by SEQ ID NO. 1, and have the amino acid sequence shown in SEQ ID NO. 2.
  • Another object of the present invention is to provide a recombinant vector containing the novel gene of the present invention.
  • a recombinant vector containing the nucleotide sequence shown in SEQ ID NO. 1 is constructed.
  • Yet another object of the present invention is to provide a method for preparing a recombinant hepatitis B surface antigen fusion protein, by transforming a host cell with the recombinant vector to obtain a transformant, and further culturing the transformant to prepare a recombinant antigen.
  • a novel gene of the present invention is constructed by a genetic engineering method, and a corresponding primer (SEQ ID NO. 3 ⁇ 6) is designed and synthesized based on the gene sequence of hepatitis B virus (adr subtype) to contain hepatitis B
  • a corresponding primer SEQ ID NO. 3 ⁇ 6
  • the plasmid of the full-length gene of the virus was used as a template, and the gene sequences corresponding to the hepatitis B surface antigen S and preS2 (Met ⁇ Gly 26 ) were synthesized by PCR, and the preS2 (Met ⁇ Gly 26 ) sequence was fused to the 3, end of the S sequence.
  • a novel hepatitis B surface antigen fusion gene was developed, and its sequence is shown in SEQ ID NO. 1.
  • the new DNA can be ligated with the vector to construct a recombinant vector, and then transform the host cell to prepare a recombinant protein.
  • the gene can also be directly constructed into a recombinant DNA vaccine for the prevention or treatment of hepatitis B.
  • the protein encoded by the gene of the present invention contains the amino acid sequence shown in SEQ ID NO. 2, which is a new hepatitis B virus surface antigen (fusion antigen) having both S and pre-S2 epitopes.
  • SEQ ID NO. 2 is a new hepatitis B virus surface antigen (fusion antigen) having both S and pre-S2 epitopes.
  • the constructed fusion gene placed the nucleotide sequence encoding the pre-S2 at the 3 'end of the nucleotide sequence encoding the S, so that only one translation product was generated during translation, effectively increasing the content of the pre-S2 antigen.
  • the fusion antigen has better stability than natural proteins.
  • the fusion antigen can be assembled into a spherical structure similar to the natural hepatitis B surface antigen, which has an important role in maintaining its immunogenicity.
  • the resulting fusion antigen can be isolated and purified to prepare a recombinant hepatitis B vaccine.
  • the protein of the present invention also includes those amino acid sequences that are substantially the same as SEQ ID N0.2, or degenerate amino acid sequences derived from it, and their biologically active analogs, These homologues have substituted, deleted, extended, substituted and even modified sequences based on SEQ ID NO. 2, and the proteins encoded by these sequences are substantially the same as those encoded by SEQ ID NO. 2. Biological activity and function.
  • a recombinant vector containing the nucleotide sequence of the present invention for example, the sequence of SEQ ID NO. 1 and a host cell transformed with the recombinant vector are provided.
  • the gene of the present invention can be inserted into various vectors by a method of inserting after digestion to construct a recombinant vector.
  • the synthetic gene of SEQ ID NO. 1 is inserted into the pA0815 vector after enzymatic digestion to construct a recombinant vector pA08i5-SS2.
  • the recombinant vector of the present invention can be transformed into host cells to obtain transformants that can be cultured and expressed on a large scale.
  • Cultured transformants can obtain expressed recombinant proteins. Can be used to prepare recombinant hepatitis B vaccine. Host cells that can be used include, but are not limited to, prokaryotic hosts such as E. coli, and eukaryotic hosts such as yeast, insect, plant, and mammalian cells. In one embodiment of the present invention, the recombinant vector constructed according to the present invention is transformed into a Pichia pastoris strain GS115 to obtain a transformant.
  • the fusion antigen has the complete structure of S + preS2 (Met ⁇ Gly 26 ), and has better stability than natural proteins.
  • Figure 1 shows the structure of the SS2 fusion gene.
  • Figure 2 shows the structure of the recombinant vector pA0815-SS2.
  • Figure 3 shows the results of Western blot analysis of the SS2 fusion antigen.
  • A detect with S antibody
  • B detect with pre-S2 antibody.
  • the left band in the figure is the pre-stained protein molecular weight standard (20.0kd, 26.0kd, 33.0kd, 50.0kd, 85.0kd, and 118.0M from bottom to top), and the right is the blotting band.
  • FIG. 4 shows the SDS-PAGE electrophoresis of the purified antigen.
  • the left band in the figure is the protein molecular weight standard (from bottom to top: 14.4kd, 18.4kd, 25.0kd, 35.0kd 45.0kd, 66.2kd, and 116.0kd), and the right is the antigen staining band.
  • Figure 5 is an electron micrograph of the fusion antigen particles.
  • Example 1 Synthesis of a hepatitis B surface antigen fusion gene with a pre-S2 epitope at the carboxy terminus and construction of an expression vector
  • Pichia strain GS115 His', Mut +
  • E. coli ToplOF and Pichia expression plasmid pA0815 were purchased from Invitrogen, E. coli DH5 ⁇ and cloning vector pBluescript II SK (+)
  • Restriction enzymes and Taq DNA polymerase were purchased from NEB, calf intestinal alkaline phosphatase (CIP), T4 DNA ligase was purchased from MBI, and DNA fragment recovery kit was purchased from Boehringer Mannheim.
  • each of the P1 and P4 primers contains an EcoR I site outside the coding region.
  • a plasmid containing the full-length gene fragment of hepatitis B surface antigen (Qi Zuhe, Yan Jun, Xiong Weijun, et al. Determination of the full sequence of hepatitis B virus adrNC-1 DNA.
  • pHBV pHBV was used as the substrate
  • P1 and P2 were used as primers to synthesize the S fragment
  • P3 and P4 were used as primers to synthesize the pre-S2 fragment
  • the product of the above reaction was used as the substrate
  • P1 and P4 were used as primers to synthesize the SS2 fusion gene fragment.
  • the specific reaction conditions are as follows:
  • the synthesis conditions for S and pre-S2 fragments are: 94 ° C 3min (minutes); 94 ° C 30s (seconds), 45 ° C 45s, 72
  • the PCR reaction conditions are as follows: 94 ° C 3min; 94 ° C 30s, 55 ° C 45s, 72 ° C 70s,
  • Figure 1 shows the structure of the SS2 fusion gene fragment.
  • the PCR product was digested with EcoR I and ligated with pBluescript II SK (+) vector linearized with EcoR I and treated with dephosphorylase (CIP) to obtain a recombinant vector (pBlue-SS2), which was transformed into E. coli DH5 ⁇ for plasmid preparation.
  • the EcoR I digestion map was analyzed, and the recombinants inserted into a single fragment were selected and commissioned by Shanghai Ji Kang Biotechnology Co., Ltd. for sequence determination.
  • the sequence of the constructed SS2 fusion gene fragment is as follows: SEQ ID NO. 3
  • the pBluescript II SK (+) vector inserted with the SS2 fragment was digested with EcoR I, the insert was separated, ligated to the pA0815 vector linearized with EcoR I and treated with CIP alkaline phosphatase, transformed into E. coli ToplO F ', and a single colony was prepared
  • the plasmid was digested with A ⁇ ⁇ and BamH I, and the digestion map was analyzed.
  • a single forward inserted recombinant was selected to construct a successful expression vector pA0815-SS2 containing the SS2 fusion gene.
  • the structure of the vector is shown in FIG. 2.
  • Hepatitis B surface antigen reverse-phase hemagglutination (RPHA) kit and surface antigen (S) enzyme labeling kit were purchased from Shanghai Kehua Biotechnology Co., Ltd. Hepatitis B surface antigen S and pre-S2 antibodies were obtained from Oxford Biotechnology Limited and Orbigen Inc. .company's product.
  • the plasmid pA0815-SS2 was linearized with Bgl ll, extracted with phenol / chloroform, and precipitated with alcohol and electroporated.
  • GS115 cells were transformed. With MD [1.34% YNB (without amino acids), 4X 10- 5% biotin, 2% glucose] agar plates and positive clones were screened. Because the GS115 strain itself cannot synthesize histidine, only strains incorporating the pA0815-SS2 vector sequence containing the HIS4 gene can grow on histidine-free media.
  • His + picked single colonies were inoculated in 100ml MGY medium (1.34% YB, 1% glycerol, 4X 10- 5% biotin) 30 ° C overnight shaking culture, cells were harvested, suspended in 20ml MM medium (1.34 % YNB, 4X 1 (T 5 % biotin, 0.5% methanol) continued to culture. 100 ⁇ l of methanol was added every 24 hrs and samples were taken. The cells were collected by centrifugation and induced for a total of 72 hrs.
  • RPHA reverse antigen hemagglutination method
  • the crude antigen extract was subjected to reduced SDS-PAGE electrophoresis and transferred to a nitrocellulose membrane.
  • S and pre-S2 antibodies were used as primary antibodies, and alkaline phosphatase (AP) -labeled goat anti-rabbit IgG was used as enzyme-labeled two.
  • Antibodies were performed using NBT / BCIP as a chromogenic substrate. It can be seen from FIG. 3 that the expression product can bind to both the S antibody and the pre-S2 antibody.
  • the S and pre-S2 antibody imprinted bands are in the same position, which indicates that the front S2 region has not been degraded, otherwise the S-antibody imprints will have smaller bands than the previous S2 antibody imprints.
  • the SS2 fusion antigen can form dimers and multimers, and there are corresponding color bands in the figure.
  • MGY medium 1.34% yeast nitrogen-containing basic medium, 1% glycerol, 4X 10_ 5% biotin; basal salt solution:
  • the purified fusion antigen was negatively stained with 2% phosphotungstic acid and observed by transmission electron microscope, it can be seen that the purified antigen can be assembled into spherical particles with a diameter of 20-35 nm, as shown in FIG. 5.
  • a fusion protein particle having both S and pre-S2 antigenicity can be expressed.
  • the fusion antigen can recognize both the S antibody and the pre-S2 antibody, and the chromotypes of the two are consistent, indicating that all translation products contain the pre-S2 component.
  • the novel fusion antigen according to the present invention can be assembled into spherical particles similar to the natural hepatitis B surface antigen, and has better stability than natural mid-proteins. These characteristics provide a good opportunity for the development of a cheap and effective new generation of hepatitis B vaccine. conditions of.

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Abstract

L'invention concerne un gène de fusion codant un nouveau type d'antigène de surface du virus de l'hépatite B et la protéine de celui-ci. La séquence nucléotidique, codant l'antigène preS2 du virus de l'hépatite B (Met1-Gly26) contenant des épitopes de lymphocytes B et de lymphocytes T ainsi qu'un site de liaison d'albumine polymérisée, est fusionnée à la terminaison 3' du gène de la protéine principale S antigène de surface du virus de l'hépatite B, puis l'expression est effectuée dans un système d'expression de levure. Les produits exprimés, possédant simultanément l'antigénicité de S et preS2, sont assemblés en des particules de type virus avec un diamètre d'environ 20-35nm et sont potentiellement destinés au développement de nouveaux vaccins contre l'hépatite B.
PCT/CN2005/000737 2004-05-31 2005-05-27 Gene de fusion d'un antigene de surface de l'hepatite b avec une extremite carboxyle possedant un determinant immune pres2 et proteine de celui-ci WO2005116218A1 (fr)

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CNA2004100226678A CN1704475A (zh) 2004-05-31 2004-05-31 羧端含前s2免疫决定簇的乙肝表面抗原融合基因及蛋白质

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5670630A (en) * 1988-05-13 1997-09-23 Research Corporation Technologies, Inc. Expression of hepatitis B S and preS2 proteins in methylotrophic
CN1059927C (zh) * 1994-03-10 2000-12-27 中国科学院上海生物化学研究所 羧端带有前表面抗原1抗原决定簇的乙肝表面抗原融合基因及其编码蛋白
US6635624B1 (en) * 1993-10-22 2003-10-21 Institut Pasteur Nucleotide vector composition containing such vector and vaccine for immunization against hepatitis

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5670630A (en) * 1988-05-13 1997-09-23 Research Corporation Technologies, Inc. Expression of hepatitis B S and preS2 proteins in methylotrophic
US6635624B1 (en) * 1993-10-22 2003-10-21 Institut Pasteur Nucleotide vector composition containing such vector and vaccine for immunization against hepatitis
CN1059927C (zh) * 1994-03-10 2000-12-27 中国科学院上海生物化学研究所 羧端带有前表面抗原1抗原决定簇的乙肝表面抗原融合基因及其编码蛋白

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KEITH E. ET AL, GENE, no. 67, 1988, pages 229 - 245 *
YUICHIRO O. ET AL, IMMUNOLOGY, no. 103, 2001, pages 90 - 97 *

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CN1820075A (zh) 2006-08-16
CN1704475A (zh) 2005-12-07

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