WO1986007384A1 - Procede de preparation de l'antigene p31 de surface du virus de l'hepatite b - Google Patents
Procede de preparation de l'antigene p31 de surface du virus de l'hepatite b Download PDFInfo
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- WO1986007384A1 WO1986007384A1 PCT/JP1985/000306 JP8500306W WO8607384A1 WO 1986007384 A1 WO1986007384 A1 WO 1986007384A1 JP 8500306 W JP8500306 W JP 8500306W WO 8607384 A1 WO8607384 A1 WO 8607384A1
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- Prior art keywords
- hepatitis
- protein
- surface antigen
- virus
- acid
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- 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
- A61K38/00—Medicinal preparations containing peptides
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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
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the present invention relates to a method for producing hepatitis B virus surface antigen P31. More specifically, the present invention relates to a method for producing hepatitis B virus surface antigen P31 by genetic recombination technology.
- Hepatitis B is a viral disease that occurs frequently, especially in mature zone Africa, Southeast Asia and the Far East, and has been epidemiologically suggested to cause chronic hepatitis, cirrhosis, and even primary liver cancer.
- the etiology is hepatitis B virus (HBV), a type of DNA virus, which is a 42-nm-diameter spherical particle bearing the name of the discoverer, Dane. Called a child.
- HBV surface antigen hereinafter abbreviated as HBsAg
- HBsAg HBV surface antigen
- HBsAg is limited to being obtained from the blood of human infected individuals, and even if the obtained small particles are used as materials for diagnostic reagents, they cannot be used for mass production of vaccines. It is enough.
- HBsAg gene HBsAg structural gene
- P-II molecular weight 25-29.5 kilodaltons
- P31 protein molecular weight: 31 kilodaltons
- P35 protein a molecular weight of 35 kilodaltons as a complex protein with P3U sucrose bound
- P31 has 55 amino acid residues in the pre-S region added to the N-terminus of P-I. In this region, polymerized human serum was replaced.
- the present invention is a.
- the transformant containing A is cultured, and the hepatitis B virus surface antigen P31 is produced and accumulated in the culture, and the obtained P31-containing solution is purified by a purification step including one step of affinity chromatography.
- Another object of the present invention is to provide a method for producing a hepatitis B virus surface antigen P31 protein.
- Subtypes (adr, adw, ar, ayw) may be used. For example, they can be prepared by the following method. Plasmid pBR322—EcoRI / HBV'933 (incorporating 3.2 kb ay3 ⁇ 4H BVD ⁇ A) described in JP-A-58-94897 or Jicleic Acids Res., U, 1947 (1983). pHBV933) can be double-digested with the restriction enzymes Hpal and EcoRI to obtain a 961 bp D>: A fragment containing a part of the pre-S region. Exchange 5 ⁇ ⁇ ⁇ 3 ⁇
- DNA encoding P31 can be prepared.
- a plasmid PBR322-BamHI / HBr330 (abbreviated as pHBr330) incorporating a 3.19 kb adr-type HBV DNA described in JP-A-59-74985 or Nucleic Acids Res., U, 1747 (1983).
- DMA encoding P31 can be prepared by antagonizing the above adapter with this fragment.
- DMA shown in the nucleotide sequence order of 28 to 873 is shown in Fig. 2
- DNA encoding the adr-type HBsAgP31 is shown in Fig. 2
- DMA represented by the base sequence order of 10 to 855 is exemplified.
- the DNA encoding P1 may be derived from a virus or may be chemically synthesized.
- DMA encoding ayr-type and ayw-type HBsAgP31 can also be prepared according to the method described above. '
- the DNA encoding P31 is expressed by inserting the DNA encoding P31 into the 3 'end of the promoter region that functions in various hosts (eg, Escherichia coli, G. lactis, yeast, animal cells). Can be used to construct recombinant DNA.
- hosts eg, Escherichia coli, G. lactis, yeast, animal cells.
- the promoter region may be any region containing a site necessary for initiating mR XA synthesis by the combination of RNA polymerase.
- P31 encoding DNA can be placed at the 3 'end of a promoter region capable of functioning in Escherichia coli, and P31 can be used.
- a recombinant DNA capable of expressing the encoding DNA can be constructed.
- P31-encoding DNA is introduced into the expression vectors pTRP601 and pTRP771 described in JP-A-58-201796 by the action of T4DNA ligase.
- Escherichia coli eg, C600 strain, 294 strain, W3U0 strain, RR1 strain, PR13 strain, etc.
- the 294 strain is a known bacterium: Backman. K. et al., Proc. Natl. Acad. Sci. USA, 73, 4-4 (1974): Fermentation Research Institute (Instit)
- e for Fermentation, Osaka (sometimes abbreviated as IF ⁇ ), also referred to as IF 0-14171.
- IF ⁇ a novel strain containing D ⁇ coding for ⁇ 31 . recombinant plasmid to locate the cells supporting post that holds the DXA;.
- the following approach is used one strand of ⁇ da Puta described above, 5 'AATTCCACTGCATTGTAT 3' T4 polynucleotide ⁇ by kinase - 32 Labeled with a radioisotope using P-ATP and used as a probe, a colony-hybridization method known per se.
- the transformant thus selected is cultured in a medium known per se.
- a medium for example, L-9 broth, Penassay broth and M-9 medium containing glucose and casamino acid [Miller, J. Experiments in Molecular Genetics, 431-433 (Gold Spring Harbor Laboratory, New York, 1972)] Is mentioned.
- a drug such as 33-indolylacrylic acid can be added to make the promoter work efficiently.
- the cultivation of the transformant is usually performed at 15 to 43 ° C. or 28 to 40 ° C. for 2 to 24 hours, preferably 4 to 16 hours, and if necessary, aeration and stirring may be added.
- a yeast transformant can be prepared as follows. Escherichia coli-yeast shuttle vector YEpl3 [Broach, JR et al., Gene, 8, 121 (1979)], pSH15 and pSH19 [Harashima, S. et al., Ol. Cell. Biol., 4, 7-71 (1984) , A yeast promoter region, such as the inhibitory acid phosphatase gene promoter region [Meyhack. B. et al., EMB 0J., 6_, 675 (1982)], the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene.
- the transformant carrying the novel recombinant DNA containing the DNA encoding P31 thus obtained can be selected for phenotype, for example, ampicillin resistance.
- phenotype for example, ampicillin resistance.
- K33-7B pho80—AH22, pho8-2
- AH22 IT AH22 IT
- ' ma K33-8D
- pho80—AH22, pho8-2 trpl pho80—AH22, pho8-2 trpl
- Yeast as a host '; but not limited to these': preferably, Saccharomyces cerevisiae.
- Saccharomyces cerevisiae AH22R_ was deposited with the Fermentation Research Institute as IF 0-10134, and from September 4, 1984, was deposited with the Microbiological Institute of Technology (FRI) under the accession number FER 'IP-1 7824. Has been donated as -The obtained yeast transformant ': Well, it is cultured in a medium known per se.
- Burkholder minimal medium Bostian. K. Shira, Proc. Natl. Acad. Sci. USA, 77, 4505 980): can be mentioned.
- Culture of the yeast transformant is usually carried out at 15 to 40 ° C., preferably 24 to 37 hours, for 10 to 96 hours, preferably: 24 to 72 hours, and if necessary, aeration and stirring may be added.
- Bacillus subtilis which encodes ⁇ 3 at the 3 ′ end of a promoter region which can function in animal cells, is known per se.
- P31 can be produced by transforming a host with the recombinant D ⁇ ′ ⁇ and culturing the transformant according to the method described above, but Escherichia coli and yeast are more preferred as the host.
- the resulting P31 may be glycosylated or glycosylated.
- P31 obtained from a transformant of Escherichia coli is not substantially glycosylated, but one molecule of P31 may have a sugar bound thereto.
- a transformant containing HBsAgDNA inhibits the growth of the transformant itself by the production of a surface antigen gene product.
- the use of a DNA that eliminates growth will eliminate growth inhibition and increase the production of P31.
- the P31 activity of the product can be determined by, for example, binding the sample to cellulose activated with bromo cyanide ⁇ paper and reacting it with 125 I-anti-HBsAg antibody from Austria ⁇ —125 (Dynabot). It can be measured by the method [Fuj i sawa, Y., ucleic Acids Res., 11, 3581 (1983)].
- the cells are collected by a known method.
- the cells are suspended in a buffer containing a protein denaturant such as urea or guanidine hydrochloride, and stirred in a cold place.
- a method for obtaining a supernatant containing P31 by centrifugation, or by suspending in a buffer, disrupting the cells by sonication, lysozyme and / or freeze-thawing, and then removing P31 by centrifugation A method of obtaining a supernatant containing the urea may be used as appropriate.For example, the cells are collected, suspended in a buffer, lysozyme is added, homogenized, lysed, and then a buffer containing urea (3 to 10 M) is added. After stirring (0.5 to 8 hours at 0 to 10 hours), a method of obtaining a supernatant by eccentric separation is preferable.
- P31 can be more efficiently extracted by adding a surfactant such as Triton X-100.
- a surfactant such as Triton X-100.
- Dexcholate or a protein denaturing agent such as guanidine hydrochloride. Separation and purification of the P31 protein from the above-mentioned extract are performed by a purification process including affinity chromatography.
- poly-HSA as a ligand, affinity chromatography, and antibody column treatment using an antibody against HBsAg, especially a monoclonal antibody.
- formylcelloline Seikagaku Corporation
- Affigel-1 15 Bio-Rad Co., Ltd.
- formyl cell mouth fine is particularly preferable.
- Poly-HSA can be produced by polymerizing human serum albumin using a cross-linking agent (glutar aldehyde if it is weak).
- the P31-containing solution (cellular supernatant) was added to a buffer solution [phosphate buffer, etc .: Adsorb to the above column equilibrated with, and elute with buffer.
- a surfactant such as Tween 20
- urine protein denaturant
- the eluted fraction containing the P31 protein is collected and, if desired, concentrated by ultrafiltration or the like.
- the self-concentrated solution is desirably reduced using an SH reagent such as dithiothreitol, and then further purified using a high-performance liquid chromatography using a reversed-phase column or a hydrophobic force column such as hydrophobic chromatography.
- an oral chromatography process Preferably, it is subjected to an oral chromatography process.
- Carriers for the high-performance liquid outlet Matogurafi one, alkyl (C: - 1 about 8) of gay-containing ones e.g., AP- 202 300A (C 8), AP- 224 300A (C 8) (YMC ShimaHisa ), Ultrapore RPSC (Beckman). Hypore RP304 (Biorad), among which — 202 300A (C 8 ) and AP-224 300 C 8 ) are preferred, and AP — 224 300A (C) 8 ) is preferred.
- the Hydro Ho Big chromatographic carriers as are alkyl Le (C t - about 18) of the carriers include for example Butyl Toyopearl (Toyo Soda Manufacturing), O lipped Le Sepharose (Fuarumashia Co., Ltd.).
- high performance liquid chromatography using a reversed phase column is advantageously used.
- the high-performance liquid chromatography using the reversed-phase ram described above can be performed, for example, by dissolving water, a lower alcohol such as C (ethanol, propanol, etc.), acetonitrile, etc. as an elution solvent with trifluoroacetic acid or the like at pH 1.2.
- the dissolution rate is preferably adjusted to ⁇ 5.0, and the dissolution rate is preferably 0.1 to 100 ml / min, more preferably 0.5 to 30 tnl, / min.
- the obtained fraction containing the P31 protein can be subjected to freeze-drying as desired by Jf to give a white powder.
- a sample is reacted with a plastic plate coated with poly-HSA, and then the P31 protein bound to poly-HSA is subjected to horseradish.
- Enzyme immunoassay (EUSA) method using anti-HBsAg monoclonal antibody conjugated to peroxidase (HRP) or P31 protein conjugated to poly-HSA can be used in Austria! !
- the radioimmunoassay (RIA) method can be used, which uses 125 I-anti-HBsAg antibody (125 Dynabot).
- HBsAgP31 protein having the following properties can be obtained.
- the substantially pure P31 protein produced by the method of the present invention has the same biological activity as HBsAg small particles produced from blood of known HB.V-infected persons, It can be used as a vaccine for diagnosis, prevention and treatment of HBV in the same manner as HBsAg small abductor.
- the activity (specific activity) of the P31 protein for determining the protein purity was measured by ELISA. That is, a wave test solution (00 z J2 of F3L protein-containing solution) was added to imnoplate ⁇ (manufactured by Nunc) to which poly-HSA obtained in 1 of Reference Example 4 was physically adsorbed beforehand, and reacted at 4 ° C for 1 ⁇ . Then, the plate was washed with PBS containing 5% serum and 0.05% Tween 20, and 100 ⁇ J2 of an anti-HBsAg monoclonal antibody solution conjugated with hosradish peroxidase was added to each well of the plate.
- the plate was washed again with the above buffer solution, and then mixed with 0-phenylenediamine (4 mg, 80 ml) and aqueous hydrogen peroxide (4i2 / 10 ml).
- An acid-phosphoric acid buffer solution pH 5.0 (100, u ⁇ ) was added, and the mixture was allowed to react at room temperature for 30 minutes.2 'Sulfuric acid (50 J2) was added to stop the enzyme reaction.
- FIG. 1 shows the DNA sequence encoding adr-type HBsAgP31 (upper row) and the corresponding amino acid sequence (lower row).
- FIG. 2 shows the DNA sequence encoding adw-type HBsAgP31 (top) and the corresponding amino acid sequence (bottom).
- FIG. 3 shows a construction diagram of the plasmid ⁇ -1, where the symbols E, S, B, H and X represent EcoRI, Sail, BamHl, HindHI and Xhoi, respectively.
- FIG. 4 shows the construction diagram of plasmid PPKT700-1, and the symbols E.S, ⁇ , ⁇ and X represent EcoRI, Sail, BamHI, Hind ⁇ and Xhol, respectively.
- FIG. 5 shows a construction diagram of the plasmid PGLD906-1. The symbols E, S, B, H and X represent EcoRI, Sail, BamHI, Hind IE and Xhol, respectively.
- FIG. 3 shows a construction diagram of the plasmid ⁇ -1, where the symbols E, S, B, H and X represent EcoRI, Sail, BamHl, HindHI and Xhoi, respectively.
- FIG. 4 shows the construction diagram of plasmid PPKT700-1, and the symbols E.S,
- FIG. 6 shows a construction diagram of the expression plasmid pTRP P31-R of adr-type HBsAgP31 for Escherichia coli, where symbols E, B, C and P represent EcoRI, BamHI, Clal and Pstl, respectively.
- FIG. 7 shows a construction diagram of the expression plasmid pTRP P31-W2 of the adw-type HBsAgP3 ⁇ for Escherichia coli, where the symbols E, B, C and P represent EcoR and BamHI, Clal and Psti, respectively.
- FIG. 8 shows a construction diagram of the expression plasmids pGLD P31-R, PH0P31-R and pPKT ⁇ 31—R of the adr-type HBsAgP31 for yeast, wherein the symbols ⁇ , ⁇ , S, ⁇ , X and C are These represent EcoRI, BamHI, Sail, Hindi, Xhol and Clal, respectively.
- FIGS. 9 and 10 show construction diagrams of the expression plasmid pPHOP31-W for adw-type HBsHgP31 for yeast, and the symbols EP, P, B, C, S and H are EcoRI, Pstl, BamHI, Clal, respectively. , Sail and HindlE.
- FIG. 11 shows the results of SDS-polyacrylamide slab gel electrophoresis in (1) and (2) described after Example 7.
- Each yeast transformant containing the P31 gene expression plasmid obtained in Examples 8 and 9 was cultured in Burkholder and its low-phosphate medium at 30 ° C for 2 days. Washed with saline.
- Miyanohara.A. Et al. [Supply the cells by Zymolyase [manufactured by Seikagaku Kogyo Co., Ltd.] according to the method described above, and then add 0.1% Triton X-100 to the bodyoblast. ⁇ 31 were extracted. The lysate was subjected to eccentric separation at room temperature at 15,000 rpm for 15 minutes to obtain a supernatant. The P31 activity of the supernatant was measured using Ausim® [Apot Co., Ltd.]. The results are shown in Table 6, where the production of P31 was calculated as per J2 of broth.
- P3L was measured with Auszyme II using HBsAg particles as a standard.
- Plasmid was extracted from the 294 strain, and Escherichia coli C600 / pTRP P31-R and Escherichia coli C600 / pTRP P31-W2 were obtained by transforming Escherichia coli C600 ⁇ using the plasmid.
- the cells were suspended in a lysate containing lQmM Tris-HCl (pH 8.0), 5m EDTA, 1mM phenylmethylsulfonylfluoride, and 5mg / ml lysozyme, and lysed. Guanidine hydrochloride was added to the lysate to a final concentration of 7M and incubated at 37 ° C for 2 hours. The lysate was centrifuged at room temperature at 15,00 Qrpm for 15 minutes to obtain a supernatant. The P31 activity of the supernatant was measured by the direct immunoassay described above. The results are shown in Table 7, where the amount of P31 produced was calculated per ml of broth.
- One unit of HBsAg is a value of the number of force of the 125 I-anti-HBsAg antibody attached to an Austri-125 kit that binds to 1 ng of HBsAg small particles.
- Example 11 Using Escherichia coli C600 / pTRP P31-R obtained in Example 11, the eluate concentrate 0.6ml (2.1 X 10 6 Yuni' from obtained poly- HSA-cell outlet Fine force ram in Example 7 according to the method described 6 ml of DTT was added to the mixture, and the mixture was reduced at 37 ° C. for 1 hour, followed by filtration using a membrane filter. 500 ⁇ J2 of the filtrate was adsorbed on an AP224 300 C 8 ) reversed-phase column (YMC Shimakusha), and trifluoric acid-acetonitrile-n-propyl alcohol was used as the elution solvent. High performance liquid mouth chromatography was performed.
- the substantially pure P31 protein (adr type) produced by the above method had the same properties as the P31 protein (adr type) obtained in Example 7.
- Cystine and cystine were subjected to formate oxidation of the F31 protein according to the Haas method, then hydrolyzed in a constant boiling point hydrochloric acid under reduced pressure for 24 hours, and quantified as cystinic acid by an amino acid analyzer.
- Threonine, serine and tributophane were determined by hydrolyzing ⁇ 31 protein in constant boiling hydrochloric acid containing 4% thioglycolic acid under reduced pressure for 16, 20, 24 and 30 hours. Weighed. Amino acid amino acid values were determined by averaging the values obtained after 25, 50 and 180 minutes of hydrolysis. However, tryptophan was obtained by hydrolysis for 24 hours, and serine and threonine values were obtained by extrapolating the hydrolysis time to 0 hours. Table 8 shows the results.
- Table 9 shows the deposits of each microorganism with the depository organizations and their accession numbers. Replacement In the table, IF 0 represents the Fermentation Research Institute, FRI represents the Microbial Industrial Technology Research Institute of the Ministry of International Trade and Industry of Japan, E. coli represents Escherichia coli, and S. cerevisiae represents Saccharomyces cerevis iae3 ⁇ 4r.
- E. coli 294 is publicly known [Backraan.k. Et al., Proc. Xatl. Acad. Sci. USA, 13, 4174 (1974):
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Abstract
On prépare l'antigène P31 de surface du virus de l'hépatite B en cultivant un transformant contenant de l'ADN recombinant préparé en introduisant de l'ADN de codage de P31 dans le terminal 3' d'une région de promotion.
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/JP1985/000306 WO1986007384A1 (fr) | 1985-06-03 | 1985-06-03 | Procede de preparation de l'antigene p31 de surface du virus de l'hepatite b |
EP85304735A EP0171908A3 (fr) | 1984-07-11 | 1985-07-03 | Antigène de surface du virus de l'hépatite B et production |
EP19900202097 EP0401941A3 (fr) | 1984-07-11 | 1985-07-03 | Antigène de surface du virus de l'hépatite B, et sa production |
PH32500A PH22118A (en) | 1984-12-12 | 1985-07-09 | Method for producing a hepatitis b virus surface antigen |
JP60153238A JPH082306B2 (ja) | 1984-07-11 | 1985-07-10 | B型肝炎ウイルス表面抗原およびその製造法 |
CN85106190A CN85106190A (zh) | 1984-07-11 | 1985-07-12 | 乙型肝炎病毒表面抗原的生产 |
JP5113354A JPH0690781A (ja) | 1984-07-11 | 1993-05-14 | B型肝炎ウイルス表面抗原の製造法 |
Applications Claiming Priority (1)
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PCT/JP1985/000306 WO1986007384A1 (fr) | 1985-06-03 | 1985-06-03 | Procede de preparation de l'antigene p31 de surface du virus de l'hepatite b |
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WO1986007384A1 true WO1986007384A1 (fr) | 1986-12-18 |
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PCT/JP1985/000306 WO1986007384A1 (fr) | 1984-07-11 | 1985-06-03 | Procede de preparation de l'antigene p31 de surface du virus de l'hepatite b |
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5971694A (ja) * | 1982-09-08 | 1984-04-23 | スミスクライン・バイオロジカルス・ソシエテ・アノニム | 肝炎b型ウイルスワクチン |
JPS5974991A (ja) * | 1982-09-09 | 1984-04-27 | マツクス・プランク・ゲゼルシヤフト・ツア・フエルデルング・デア・ヴイツセンシヤフテン・エ−・フアウ | B型肝炎細胞表面抗原を生産するための組換えdna分子 |
-
1985
- 1985-06-03 WO PCT/JP1985/000306 patent/WO1986007384A1/fr unknown
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5971694A (ja) * | 1982-09-08 | 1984-04-23 | スミスクライン・バイオロジカルス・ソシエテ・アノニム | 肝炎b型ウイルスワクチン |
JPS5974991A (ja) * | 1982-09-09 | 1984-04-27 | マツクス・プランク・ゲゼルシヤフト・ツア・フエルデルング・デア・ヴイツセンシヤフテン・エ−・フアウ | B型肝炎細胞表面抗原を生産するための組換えdna分子 |
Non-Patent Citations (1)
Title |
---|
Natare, Vol. 298, No. 5872 (July, 1982) Pablo Valenzuela, et al: "Sythesis and assembly of hepatitis B virus surface antigen particles in yeast" P.347-350 * |
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