CN1704475A - HBsAg fusion gene with carboxyl-end containing pre S2 immune determinant and protein thereof - Google Patents
HBsAg fusion gene with carboxyl-end containing pre S2 immune determinant and protein thereof Download PDFInfo
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- CN1704475A CN1704475A CNA2004100226678A CN200410022667A CN1704475A CN 1704475 A CN1704475 A CN 1704475A CN A2004100226678 A CNA2004100226678 A CN A2004100226678A CN 200410022667 A CN200410022667 A CN 200410022667A CN 1704475 A CN1704475 A CN 1704475A
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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
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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
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- 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
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- 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
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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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5258—Virus-like particles
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- 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
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- 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
Abstract
The invention relates to the genes and proteins encoding novel hepatitis B virus surface antigen, wherein the pro-S2 antigen (Metl-Gly26) coded sequence of the hepatitis B virus and having B cell and T cell and polymeried albumin receptor binding site is fused onto the end of the main protein (S) gene and expressed in a yeast system, the expression product has both S and preS2 antigen property, and can be assembled into virus sample particles with a diameter of 20-35 nm.
Description
Technical field
The present invention relates to a kind of new hepatitis B surface antigen encoding gene, relate in particular to the hepatitis B surface antigen blending gene of the carboxyl terminal containing precursor 32 immune decision cluster that makes up by gene engineering method.
The invention still further relates to the recombinant vectors and the encoded protein matter thereof of this gene.
Background technology
Hepatitis B is a kind of communicable disease with serious harm that is caused by hepatitis B virus, extensively distributes in the whole world, and according to estimates, only just have an appointment 1.2 hundred million hepatitis B virus carriers of China, wherein the patient has 10,000,000 people approximately.At present hepatitis B also being lacked effective methods of treatment, is prevention and the effective approach of control hepatitis B and carry out the hepatitis b vaccine immune inoculation.
Early stage Hepatitis B virus vaccine isolating hepatitis B surface antigen particle from hepatitis B virus healthy carrier blood plasma is made, because the blood plasma source is limited, and the consideration of cost and security aspect in addition, this class vaccine is stopped using in China.The recombination engineered vaccine by hepatitis B virus surface antigen master albumen (S) formation of domestic and international widely used recombination yeast or Chinese hamster ovary celI generation at present.Compare with vaccine from blood, the recombinant vaccine cost is lower, and security is better, has obtained good immune effect.But simultaneously the recombiant vaccine of this single S also has many deficiencies, and is long as immune programme for children, immunologic escape that the hepatitis B virus mutant strain causes and part crowd no response or low reply etc.Therefore, exploitation immunogenicity new generation vaccine better, that price is more cheap still has its urgency.
Hepatitis-B virus cytomembrane albumen contains (L) greatly, in (M) and three kinds of compositions of main albumen (S), wherein S albumen is made up of 226 amino acid, before S albumen, distinguish and distinguish by a preceding S1 (preS1) who forms by 119 amino acid by the preceding S2 (preS2) that 55 amino acid are formed, wherein, albumen during PreS2 and S constitute together, preS1 and M constitute large protein together.There are some researches show, preS2 antigen has the immunogenicity stronger than S albumen, with the hepatitis B surface antigen immune mouse that contains preS2 and S district simultaneously, can allow the unresponsive mouse of S antigen is produced antibody response (Milich DR., Thornton GB., Neurath AR., et al.Enhancedimmunogenicity of the preS region of hepatitis B surface antigen.Science1985,228:1195-1199).Therefore, in vaccine, add the preS2 antigenic component and may further strengthen its immune effect.Because preS2 antigen contains the responsive site of a plurality of proteolytic enzyme, particularly its N holds peptide bond between the 48th arginine and 49 Threonines to be vulnerable to the effect of proteolytic enzyme and (the Langley KE. that ruptures, Egan KM., Barendt JM., et al.Characterization of purified hepatitis B surface antigen containingpre-S (2) epitopes expressed in saccharomyces cerevisiae.Gene 1988,67:229-245), cause preS2 antigen in the process of expressing and preparing, to be easy to degraded and inactivation.Synthetic preS2 polypeptide confirms, its aminoterminal 26 amino acid (Met
1-Gly
26) be the main antibody combining site in this zone, antibody that should synthetic polypeptide can in and virulence (the Neurath AR. of hepatitis B virus, Kent SBH., Parker K, et al.Antibodies to a syntheticpeptide from the preS120-145 region of the hepatitis B virus envelope arevirus neutralizing.Vaccine 1986,4:35-37).Because preS2 and S gene contain translation initiation site separately, the structure of preS2+S is when carrying out vivoexpression, two kinds of products of albumen and main albumen in will producing, thereby reduced the content (HuiJ of preS 2 antigen in the product, Li G, Kong Y et al. Expression and Characterization of chimerichepatitis B surface antigen particles carrying preS epitopes.Journal ofBiotechnoly, 1999,72:49-59; Chinese patent CN 1067721C; Chinese patent CN1058525C).
Summary of the invention
One object of the present invention is to provide a kind of gene of new coding hepatitis B virus surface antigen, the nucleotide sequence that contains proteic nucleotide sequence of coding hepatitis B surface antigen S and coding hepatitis B surface antigen pre-s2 protein, the nucleotide sequence of 1-26 amino acids of described pre-s2 protein of it is characterized in that encoding is connected in 3 ' end of the proteic nucleotide sequence of the described S of coding, has made up the new gene of the sequence shown in SEQ ID NO.1 in a specific embodiments of the present invention.
A further object of the present invention is to provide a kind of protein with hepatitis B virus surface antigen S and preS 2 antigen, this protein comprises the aminoacid sequence by new coded by said gene of the present invention, in a specific embodiment, the present invention has the albumen of hepatitis B virus surface antigen S and preS 2 antigen by SEQ ID NO.1 coding, has the aminoacid sequence shown in the SEQ ID NO.2.
Another object of the present invention is to provide the recombinant vectors that contains the new gene of the present invention, in a specific embodiment of the present invention, made up the recombinant vectors that contains nucleotide sequence shown in the SEQ ID NO.1.
A further object of the present invention is to provide the preparation method of recombination hepatitis B surface antigen fusion rotein, by with described recombinant vectors transfection host cell, obtains transformant, further cultivates this transformant and prepares recombinant antigen.
According to an aspect of the present invention, made up new gene of the present invention by engineered method, gene order according to hepatitis B virus (adr hypotype) designs and synthesizes corresponding primer (SEQID NO.3～6), with the plasmid that contains the hepatitis B virus full-length gene is template, has synthesized hepatitis B surface antigen S and preS2 (Met by pcr amplification
1-Gly
26) pairing gene order, and with preS2 (Met
1-Gly
26) the sequence 3 ' end that is blended in the S sequence made up novel hepatitis B surface antigen blending gene, its sequence is shown in SEQ ID NO.1.DNA that can this is new is connected with carrier and is built into recombinant vectors, and then changes (dying) and change host cell, thus the preparation recombinant protein.Also this gene directly can be built into recombinant DNA vaccine, be used for the prevention or the treatment of hepatitis B.
According to a further aspect in the invention, albumen by genes encoding of the present invention contains just like the aminoacid sequence shown in the SEQID NO.2, it is a novel hepatitis B virus surface antigen (fused antigen) that has S and preS 2 antigen determinant simultaneously, the nucleotide sequence of S2 placed 3 ' end of the nucleotide sequence of coding S before the fusion gene that makes up will be encoded, thereby have only a kind of translation product to produce when serving as interpreter, effectively improved the content of preS 2 antigen.Simultaneously, owing to removed the responsive site of the main proteolytic enzyme in preceding S2 district, this fused antigen has than the better stability of native protein.In addition, this fused antigen can also be assembled into and the similar globosity of natural hepatitis B surface antigen, and these characteristics have vital role to its immunogenic keeping.The fused antigen that is produced can be prepared into reconstituted hepatitis B vaccine after separation and purification.
It will be understood by those skilled in the art that, protein of the present invention also comprises those and the essentially identical aminoacid sequence of SEQ ID NO.2 or comes from its aminoacid sequence of degeneracy and they are at biologically active congener, these congeners have on the basis of SEQ IDNO.2 through that replacement, disappearance, prolong, metathetical and even the sequence of modifying, the coded protein of these sequences has protein substantially identical biologic activity and the function coded with SEQ ID NO.2.
In accordance with a further aspect of the present invention, provide and contain nucleotide sequence of the present invention, for example the recombinant vectors of SEQID NO.1 sequence and by the recombinant vectors transformed host cells.The method of inserting after can cutting by enzyme is inserted gene of the present invention in the various carriers and is built into recombinant vectors.In one embodiment of the invention, the gene with synthetic SEQ ID NO.1 sequence inserts pAO815, vector construction recombinant vectors pAO815-SS2 after enzyme is cut.
When further preparing expressing protein, but the transformant that recombinant vectors transfection host cell of the present invention can be obtained large scale culturing and express, cultivate the recombinant protein that this transformant can obtain to express, to this albumen separate with purifying after promptly can be used for preparing reconstituted hepatitis B vaccine.Operable host cell includes but not limited to for example colibacillary prokaryotic hosts, and the eucaryon host of yeast, insect and mammalian cell for example.In one embodiment of the invention, the recombinant vectors that the present invention is made up changes (dye into) and changes to pichia spp (PichiaPastoris) bacterial strain GS115 and obtain transformant, the pichia pastoris engineered strain that contains this fusion gene by structure, efficiently expressed the fusion particle that has S and preS 2 antigen simultaneously, this fused antigen has S+preS2 (Met
1-Gly
26) complete structure, and have than the better stability of albumen in natural.
Description of drawings
Fig. 1 is the structural representation of SS2 fusion rotein.
Fig. 2 is the agarose gel electrophoretogram of PCR product.
1，S2；2，S；3，DNA?marker(λDNA/EcoR?I+Hind?III)；4，SS2
Fig. 3 is the restriction enzyme mapping of cloning vector.
1，DNA?marker(λDNA/EcoR?I+Hind?III)；2，pBlue/EcoR?I；
3，pBlue-SS2/EcoR?I.
Fig. 4 cuts for the enzyme of yeast expression vector pAO815-SS2 and identifies collection of illustrative plates (1% agarose gel electrophoresis).
1，DNA?marker(λDNA/EcoR?I+Hind?III)；2，pAO815；3，pAO815-SS2.。Above-mentioned two kinds of plasmids all carry out double digestion with Avr II and BamH I.
Fig. 5 is the Western blot analytical results of SS2 fused antigen.
A, the S antibody test; B detects with pre-s2 antibody; 1, protein dyes Marker in advance;
2, expression product.
Fig. 6 is the transmission electron microscope photo of SS2 fused antigen.
Embodiment
Below in conjunction with accompanying drawing,, illustrate but do not limit the present invention by detailed description to better embodiment of the present invention.
Synthetic and the sequencing of the hepatitis B surface antigen blending gene of [embodiment 1] carboxyl terminal containing precursor 32 immune decision cluster
Materials and methods
1. bacterial classification and plasmid
Pichia yeast strain GS115 (His
-, Mut
+), intestinal bacteria Top10F ' and Pichia expression plasmid pAO815 are available from Invitrogen company, and bacillus coli DH 5 alpha and cloning vector pBluescript II SK (+) they are general bacterial strain and plasmid.
2. main raw and reagent
Restriction enzyme and Taq archaeal dna polymerase are available from NEB company, and calf intestine alkaline phosphatase (CIP), T4 dna ligase are available from MBI company, and dna segment reclaims test kit available from Boehringer Mannheim company.Anti-phase blood clotting test kit is available from Lanzhou Institute of Biological Products, and S and preceding S2 enzyme are exempted from test kit respectively available from Shanghai Kehua Bio-technology Co., Ltd and hepatitis reagent development center, Beijing.
3.PCR primer is synthetic and the segmental amplification of SS1 fusion gene
According to document (outstanding ancestral and, Yan Jun, Xiong Weijun waits the complete sequence determination of hepatitis B virus adr NC-1DNA. Chinese science (B collects), 1988,12:1300-1304.) Bao Dao hepatitis B virus (adr hypotype) gene order, synthesize following four primers:
P1：5’-AAAGAATTCACCATGGAGAACACAGCATCA-3’；(SEQ?IDNO.3)
P2：5’-AATGTATACCCAAAGAC-3’；(SEQ?ID?NO.4)
P3：5’-TGGGTATACATTATGCAGTGGAACTCC-3’；(SEQ?ID?NO.5)
P4：5’-AAAGAATTCTCAGCCACCAGCAGG-3’；(SEO?ID?NO.6)
Wherein P3 primer 5 ' end has 12 bases and the complementation of P2 primer, respectively contains an EcoR I site beyond P1 and the P4 primer coding region.With contain the segmental plasmid of hepatitis B surface antigen full-length gene (outstanding ancestral and, Yan Jun, Xiong Weijun, Deng. the complete sequence determination of hepatitis B virus adr NC-1 DNA. Chinese science (B collects), 1988,12:1300-1304.) pHBV is a substrate, with P1 and P2 is the synthetic S fragment of primer, with P3 and P4 is the synthetic S2 fragment of primer, and the product with above-mentioned reaction is a substrate then, is the synthetic SS2 mosaic gene fragment of primer with P1 and P4.Concrete reaction conditions is as follows:
The segmental synthesis condition of S and S2 is: 94 ℃ of 3min (branch); 94 ℃ of 30s (second), 45 ℃ of 45s, 72 ℃ of 75s, 25 circulations; 72 ℃ of 5min.Reclaim S and S2 fragment respectively, respectively get 1ul and carry out SS2 as substrate and merge segmental amplification.The PCR reaction conditions is as follows: 94 ℃ of 3min; 94 ℃ of 30s, 55 ℃ of 45s, 72 ℃ of 70s, 25 circulations; 72 ℃ of 5min.
Fig. 1 is the segmental structural representation of SS2 mosaic gene.
4. the structure of cloning vector and gene sequencing
The PCR product is digested with EcoR I; with through EcoR I linearizing and spend pBluescript II SK (+) carrier that Starch phosphorylase (CIP) handles and is connected acquisition recombinant vectors (pBlue-SS2); transformed into escherichia coli DH5 α; carry out plasmid and prepare post analysis EcoR I restriction enzyme mapping; select and insert single segmental recon, entrust Jikang Biotechnology Co Ltd, Shanghai to carry out sequencing.The sequence of the new gene that makes up is shown in SEQ ID NO.1.
The expression of the hepatitis B surface fused antigen of [embodiment 2] carboxyl terminal containing precursor 32 immune decision cluster in pichia spp (Pichia Pastoris)
5. the structure of expression vector and evaluation
Insert SS2 segmental pBluescript II SK (+) carrier with EcoR I digestion, separate and insert fragment, with be connected through EcoR I linearizing and with the pAO815 carrier of CIP alkaline phosphatase treatment, transformed into escherichia coli Top10F ', picking list bacterium colony prepares plasmid, with Avr II and BamH I double digestion, analyze restriction enzyme mapping, select the recon that single forward inserts.
6. recombinant plasmid is to the conversion of yeast cell and the preliminary screening of positive colony
With Bgl II linearizing recombinant expression plasmid,, transform the GS115 cell with electroporation behind the alcohol precipitation through phenol/chloroform extracting.With MD[1.34%YNB (no amino acid), 4 * 10
-5The % vitamin H, 2% glucose] the agar plate screening positive clone.
7. antigenic abduction delivering and the preparation of extract just
Picking His
+Single bacterium colony, be inoculated in 100ml MGY substratum (1.34%YNB, 1% glycerine, 4 * 10
-5The % vitamin H) 30 ℃ of concussion overnight incubation, the results thalline is suspended in 20mlMM substratum (1.34%YNB, 4 * 10
-5The % vitamin H, 0.5% methyl alcohol) continue to cultivate.Every 24hr adds 100 μ l methyl alcohol and sampling, centrifugal collection thalline, coinduction 72hr.
In thalline, add an amount of broken wall damping fluid (50mM phosphoric acid buffer pH7.4,1mMPMSF, 1mM EDTA, 5% glycerine) and equal-volume granulated glass sphere (diameter 0.5mm), behind thermal agitation 30s on the vibrator, ice bath 30s, repeat 8 times, centrifugal extraction supernatant is antigen extract just.
The detection of [embodiment 3] expression product
8. the antigenicity of expression product detects
By anti-phase blood clotting method (RPHA) and ELISA first extract is carried out antigenicity and detect, working method is undertaken by the test kit specification sheets respectively.
9. the Western blot of expression product identifies
With the antigen crude extract through the SDS-PAGE electrophoresis and transfer on the nitrocellulose filter, be one anti-with anti-S of rabbit (Oxford Biotechnology Limited) and the anti-preS2 of rabbit (Orbigen Inc.) respectively, goat anti-rabbit igg with alkaline phosphatase (AP) mark is an ELIAS secondary antibody, with NBT/BCIP is chromogenic substrate, carries out engram analysis.
10. the electron microscopic observation of fused antigen
The SS2 fused antigen of purifying is carried out transmission electron microscope observing after 2% phospho-wolframic acid negative staining.
The result:
1. the segmental amplification of SS2 fusion gene
The first step PCR reaction has amplified the S (690bp) and S2 (90bp) fragment of expection size, is template with S and S2 again, is that primer carries out the PCR reaction second time with P1 and P4, has been spliced into complete SS2 (770bp) fusion gene fragment (Fig. 2).
2. the structure of cloning vector and gene sequencing
From the PCR product, reclaim the SS2 fragment,, be cloned into the RcoR I site of pBluescriptII SK (+) carrier, construction recombination plasmid with EcoR I digestion.Recombinant plasmid is through EcoR I restriction analysis, clip size and expection consistent (Fig. 3).The sequencing results shows that SS2 fusion gene fragment assembly is correct, and phase shift mutation does not take place.
3. the structure of expression vector and evaluation
To from pBluescript II SK (+) carrier, separate through the SS2 fragment that order-checking confirms, be cloned into the EcoR I site of pAO815 plasmid.With Avr II and BamH I digestion recombinant plasmid, carry out the restriction enzyme mapping analysis through agarose gel electrophoresis.Because Avr II restriction enzyme site is positioned at S gene 5 ' end (apart from about 30 Nucleotide of 5 ' end), and the pAO815 carrier contains single BamH I site, therefore recombinant plasmid can be distinguished different inserted mode (forward, reverse and multi-disc section insertion) according to the different distributions of electrophoretic band after using Avr II and BamH I double digestion.Fig. 4 is the restriction analysis figure that single forward inserts the segmental pAO815 carrier of SS1.
4. the antigenicity of the structure of expression strain and product detects
PAO815-SS2 is imported Pichia GS115 cell with electrotransformation behind Bgl II enzymolysis, with MD agar plate (no Histidine) screening His
+The transformant of phenotype.Because host bacterium itself can not be synthesized Histidine, has only the host cell of having integrated recombinant plasmid to grow.Random choose reorganization bacterium colony is made abduction delivering on a small scale, detects expression product with the anti-phase blood clotting test kit of hepatitis B surface antigen, selects the higher bacterial strain of expression amount, and further detects S and preS 2 antigen with ELISA.From 8 His
+In the transformant of phenotype, we screen a recombinant bacterial strain that can efficiently express S and preS 2 antigen, measurement result such as table 1.
The antigenicity of table 1 fused antigen SS2 detects
| RPHA | ?????????????????ELISA | |
| ??????PreS2 | ??????S | |
| 1：1024 | (2.92 1/100 dilution) | (2.03 1/10 dilution) |
5. recombinant antigen Western blot detects
Western Blot analytical results shows that expression product can also can combine with pre-s2 antibody (Fig. 5 B) with S antibody (Fig. 5 A), and the fused antigen molecular weight is about 27kd, can form dimer and part polymer.The trace result of Fig. 5 A and Fig. 5 B also further shows, the SS2 fused antigen express and leaching process in, the generation of the band of not degrading.
6. the electron microscopic observation of recombinant antigen
The SS2 recombinant antigen of purifying can be assembled into the spheroidal particle of diameter 20-35nm, as shown in Figure 6.
From The above results as can be seen, the encoding sequences of hepatitis B virus pro-S 2 antigenic determinants is blended in 3 ' end of total length S gene after, can give expression to the fusion rotein particle that has S and preS 2 antigen simultaneously.This fused antigen can be discerned S antibody under the sex change condition, also can discern pre-s2 antibody, and the colour developing banding pattern unanimity of the two shows that all translation products all contain preceding S2 composition.Novel fused antigen of the present invention can be assembled into and the similar spheroidal particle of natural hepatitis B surface antigen, and has than the better stability of albumen in natural, these characteristics for develop cheapness, Hepatitis B virus vaccine of new generation provides good condition efficiently.
Above detailed description of the present invention does not limit the present invention, and those skilled in the art can make various changes or distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the defined scope of claims of the present invention.
SEQUENCE?LISTING
＜110〉Chengdu Inst. of Biological Products
＜120〉hepatitis B surface antigen blending gene of carboxyl terminal containing precursor 32 immune decision cluster and protein
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1???????????????5???????????????????10??????????????????15
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35??????????????????40??????????????????45
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130?????????????????135?????????????????140
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<221>misc_feature
<222>(1)..(30)
＜223〉primer
<400>3
aaagaattca?ccatggagaa?cacagcatca
30
<210>4
<211>17
<212>DNA
<213>Artificial
<220>
<221>misc_feature
<222>(1)..(17)
＜223〉primer
<400>4
aatgtatacc?caaagac
17
<210>5
<211>27
<212>DNA
<213>Artificial
<220>
<221>misc_feature
<222>(1)..(27)
＜223〉primer
<400>5
tgggtataca?ttatgcagtg?gaactcc
27
<210>6
<211>24
<212>DNA
<213>Artificial
<220>
<221>misc_feature
<222>(1)..(24)
＜223〉primer
<400>6
aaagaattct?cagccaccag?cagg
24
Claims (12)
1, the gene of coding hepatitis B virus surface antigen contains the nucleotide sequence of coding hepatitis B surface antigen S proteic nucleotide sequence and coding hepatitis B surface antigen pre-s2 protein, the 1-26 amino acids (Met of the described pre-s2 protein that it is characterized in that encoding
1-Gly
26) nucleotide sequence be connected in 3 ' end of coding described S proteic nucleotide sequence,
2, the described gene of claim 1 is characterized in that, it has the described nucleotide sequence of SEQ ID NO.1.
3, a kind of hepatitis B surface antigen fusion rotein, it contains the aminoacid sequence by the described genes encoding of claim 1.
4, the described fusion rotein of claim 3 is characterized in that, contains the described aminoacid sequence of SEQ ID NO.2.
5, the recombinant vectors that contains the described nucleotide sequence of claim 1.
6, the described recombinant vectors of claim 5 is characterized in that described recombinant vectors is pAO815-SS2.
7, a kind of hepatitis B DNA vaccine, it contains the described nucleotide sequence of claim 1.
8, the described hepatitis B DNA vaccine of claim 7 is characterized in that it contains the DNA of the described sequence of SEQ IDNO.1.
9, a kind of preparation method of reconstituted hepatitis B vaccine, comprise with the described recombinant vectors transformed host cell of claim 5, obtain transformant, cultivate described transformant and obtain the recombination hepatitis B antigen coalescence protein of expressing, to this albumen separate with purifying after promptly can be used for preparing reconstituted hepatitis B vaccine.
10, the described method of claim 6 is characterized in that described host cell is a yeast.
11, the described method of claim 6 is characterized in that described host cell is intestinal bacteria, insect cell or mammalian cell.
12, a kind of reconstituted hepatitis B vaccine is characterized in that it prepares with following method:
With the described recombinant vectors transformed host cell of claim 5, obtain transformant, cultivate described transformant and obtain the recombination hepatitis B antigen coalescence protein of expressing, to this albumen separate with purifying after promptly can be used for preparing reconstituted hepatitis B vaccine.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2004100226678A CN1704475A (en) | 2004-05-31 | 2004-05-31 | HBsAg fusion gene with carboxyl-end containing pre S2 immune determinant and protein thereof |
| CNB2005800006774A CN100413970C (en) | 2004-05-31 | 2005-05-27 | Hepatitis B surface antigen blending gene and protein for carboxyl terminal containing precursor 32 immune decision cluster |
| PCT/CN2005/000737 WO2005116218A1 (en) | 2004-05-31 | 2005-05-27 | Fusion gene of hepatitis b surface antigen with carboxyl end having pres2 immune determinant and the protein thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2004100226678A CN1704475A (en) | 2004-05-31 | 2004-05-31 | HBsAg fusion gene with carboxyl-end containing pre S2 immune determinant and protein thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1704475A true CN1704475A (en) | 2005-12-07 |
Family
ID=35450897
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2004100226678A Pending CN1704475A (en) | 2004-05-31 | 2004-05-31 | HBsAg fusion gene with carboxyl-end containing pre S2 immune determinant and protein thereof |
| CNB2005800006774A Expired - Fee Related CN100413970C (en) | 2004-05-31 | 2005-05-27 | Hepatitis B surface antigen blending gene and protein for carboxyl terminal containing precursor 32 immune decision cluster |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005800006774A Expired - Fee Related CN100413970C (en) | 2004-05-31 | 2005-05-27 | Hepatitis B surface antigen blending gene and protein for carboxyl terminal containing precursor 32 immune decision cluster |
Country Status (2)
| Country | Link |
|---|---|
| CN (2) | CN1704475A (en) |
| WO (1) | WO2005116218A1 (en) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ228948A (en) * | 1988-05-13 | 1991-06-25 | Phillips Petroleum Co | Hbv particles containing s and pre-s 2 proteins and recombinant processes for their production in yeast cells |
| FR2711670B1 (en) * | 1993-10-22 | 1996-01-12 | Pasteur Institut | Nucleotide vector, composition containing it and vaccine for immunization against hepatitis. |
| CN1059927C (en) * | 1994-03-10 | 2000-12-27 | 中国科学院上海生物化学研究所 | Reconstituted hepatitis B vaccine with corboxy end having anterior surface antigen 1 determinant |
-
2004
- 2004-05-31 CN CNA2004100226678A patent/CN1704475A/en active Pending
-
2005
- 2005-05-27 CN CNB2005800006774A patent/CN100413970C/en not_active Expired - Fee Related
- 2005-05-27 WO PCT/CN2005/000737 patent/WO2005116218A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| CN1820075A (en) | 2006-08-16 |
| CN100413970C (en) | 2008-08-27 |
| WO2005116218A1 (en) | 2005-12-08 |
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