WO1986001534A1 - Adn recombinant et son utilisation - Google Patents

Adn recombinant et son utilisation Download PDF

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Publication number
WO1986001534A1
WO1986001534A1 PCT/JP1984/000423 JP8400423W WO8601534A1 WO 1986001534 A1 WO1986001534 A1 WO 1986001534A1 JP 8400423 W JP8400423 W JP 8400423W WO 8601534 A1 WO8601534 A1 WO 8601534A1
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Prior art keywords
dna
reference example
plasmid
reaction solution
fragment
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PCT/JP1984/000423
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English (en)
Japanese (ja)
Inventor
Masakazu Kikuchi
Yukio Fujisawa
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Takeda Chemical Industries, Ltd.
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Application filed by Takeda Chemical Industries, Ltd. filed Critical Takeda Chemical Industries, Ltd.
Priority to PCT/JP1984/000423 priority Critical patent/WO1986001534A1/fr
Priority to EP85304735A priority patent/EP0171908A3/fr
Priority to EP19900202097 priority patent/EP0401941A3/fr
Priority to JP60153238A priority patent/JPH082306B2/ja
Priority to CN85106190A priority patent/CN85106190A/zh
Publication of WO1986001534A1 publication Critical patent/WO1986001534A1/fr
Priority to JP5113354A priority patent/JPH0690781A/ja

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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
    • 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 novel recombinant DNA and its use. Further for more hepatitis B virus surface antigen P 3 1 co one de to DNA a promoter a region 3, the recombinant DNA obtained by ⁇ -terminated transformant and the transformant containing the recombinant DNA And producing and accumulating hepatitis B virus surface antigen P31 in the culture, and collecting the same.
  • Hepatitis B is a viral disease that occurs frequently, especially in tropical 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 spherical particle with a diameter of 42 nm and bears the name of the discoverer.
  • the outer layer has HBV surface antigens (hereinafter abbreviated as HBsAg), which are divided into subtypes such as adr, adw, ayr, and ayw depending on their antigenicity. Adw and a dr types are found in Japan.
  • HBsAg antibodies against a viral superficial antigen protect the virus from infection.
  • HBV a vaccine against hepatitis B based on HBsAg can be considered. Where power, HB
  • ⁇ V can only infect human chimpanzees, and attempts to infect cultured cells have not been successful. For this reason, HBsAg is limited to being obtained from the blood of human infected individuals, and the obtained small abductors, etc., can only be used as a diagnostic reagent material and cannot be used for production of pectin. State.
  • HBsAg gene HBsAg structural gene
  • the location and base sequence of the HBsAg gene were determined for the ayw type, which is common in Europe and the United States !: Galibert, F. et al. Nature, 2 ⁇ 1, 646 (1979); Carnay, P. et al., Nucleic Acids Res. ', 7, 335 (1979)], and its expression in Escherichia coli as a hybrid protein has been reported [Charnay, P. Edman, JC et al., Nature, 286, 893 (1980).
  • Coding the hepatitis B virus surface antigen P31 a recombinant DNA comprising a DNA inserted into the 3rd end of the promoter region,
  • Recombinant DNA comprising a DNA encoding hepatitis B virus surface antigen P31 inserted at the 3 'and 3' termini of the promoter region; I) a transformant containing NA; and
  • An object of the present invention is to provide a method for producing hepatitis B virus surface antigen P31, which comprises producing and accumulating virus surface antigen P31 and collecting the virus.
  • the NA may be of any subtype (adr, adw, ayr, ayw), for example they can be prepared by the following method. Plasmid PBR322—EcoHI / ⁇ 33C incorporating a 3.2 kb adw-type HBV DNA described in Japanese Patent Application Laid-Open No. 58-194948 or Nucleic Acids Res., Ll, 1747 U983). pHBV933 (abbreviated as pHBV933) can be double-digested with restriction enzymes Hpal and EcoRI to obtain a 961 bp DNA fragment containing a part of the pre-S region. This fragment [3 4 ⁇ ⁇ 3 ⁇ 4 ⁇ ⁇ ??
  • the DNA encoding adw HBsA P31 is represented by the nucleotide sequence of 28 to 873 of the DNA sequence shown in Fig. 1.
  • DNA mosquito, adr-type HBsAg P31 is encoded. It is shown in Figure 2 as DNA! )
  • DNAs represented by the base sequence order of 10 to 855 are listed.
  • DNA encoding P31 may be virus-derived or chemically synthesized. '' DNA_ encoding ayr-type and ayw-type HBsAg P31 is also as described above. -G-can be prepared according to the method.
  • P31 By inserting the DNA encoding P31 into the end of the promoter region that functions in various hosts (eg, Escherichia coli, Bacillus subtilis, yeast, animal cells), P31 can be introduced. It is possible to construct a recombinant DNA capable of expressing the coding DNA.
  • hosts eg, Escherichia coli, Bacillus subtilis, yeast, animal cells
  • the promoter region may be any region as long as it contains a site necessary for initiating mRNA synthesis by the binding of RNA polymerase.
  • a DNA encoding P31 when Escherichia coli is used as a host, a DNA encoding P31 can be inserted into the 3 and terminus of a promoter region that can function in Escherichia coli, and a recombinant DNA capable of expressing DNA encoding P31 can be used. s can be built. For example, P31-encoding DNA is introduced into the expression vectors pTRP-601 and pTRP771 described in JP-A-58-201979 by the action of T4 DNA ligase. I do.
  • Escherichia coli eg, C600 strain, 2904 strain, W3110 strain, RR1 strain, PR13 strain, etc.
  • Escherichia coli was purified by a known method [Cohen'S.N. Et al. Proc. Nat and Acad. Sci. USA, 69, 2110 (1972)] or a method analogous thereto.
  • the promoter to be used does not need to be limited to the trp promoter (trp-p), for example, the recA promoter (Japanese Patent Laid-Open No. 59-e509), the lac promoter, the sp promoter, etc. May be used.
  • Transformants carrying the novel recombinant plasmid DNA containing P31-encoding DNA obtained as described above include, for example, ampicillin resistance, tetracycline resistance, or resistance to both drugs. Can be selected as an expression.
  • the 294 strain is a known bacterium [Backman, K. et al., Proc. Natl. Acad. Sci. USA, 73, 4174 (197)].
  • Hmm It has also been deposited with the Institute for Fermentation, Osaka; sometimes referred to as IFO, as IO-141 171. It encodes P31 among these resistant strains
  • a new recombinant plasmid containing NA To search for a strain that harbors DNA, for example, the following technique is used: One strand of the adapter 1 described above, 5 AATTCC AC GCATTGTAT 3, and T 4 polynucleotide - labeled with a radioactive isotope ⁇ 3 ⁇ 4 by using the r _32p_ aT P by kinase, this as a Sagu ⁇ (probe), known per se of the colonies one * Haiburidize one Chillon method [Grunstein, M. and Hogness, DS, Proc ⁇ Natl. Acad. Sci. USA, 3961 (1975) makes it possible to reliably search for positively resistant clones among drug-resistant transformants already obtained.
  • the transformant thus selected is cultured in a medium known per se.
  • a medium for example, L-9 broth, Penassay (; Penassay) broth and M-9 medium containing glucose and casamino acid [Mi'ller, J., Experiments in Molecular Genetics, 431-433 C Cold Spring
  • yeast transformants can be transformed as follows. Bacteria-Yeast Shuttle 'Vector-YE pi 3 [Broach, JR et al., Gene,, 21 (1979)], pSH15 and pSHl9 [Harashima, S. et al., Mol. Cell.
  • yeast promoter region for example, an inhibitory acid phosphatase gene promoter region [Meyhack, BB, EMBOJ., 6. »675 (1982)], a glyceraldehyde 3-phosphate dehydrogenase gene promoter region! Hoi land, JP and Holland. MJ, J. Biol.
  • “Plasmid DNA was isolated from the transformants selected in this manner by an extraction method (Birnboim, HC and Doly, J., Nucleic Acids Res.,, 1513 (1979)) and used. Saccharomyces cerevisiae, such as AH22R— (leu 2 his canlcir + ph. 80 Ba Proc. Natl. Acad Sci. USA 80, 1 1983) K3 3-7 BC pho.80-AH22, pho8-2) derived from 3 or AH22R— or K33—8D (pho80—AH22, pho8-2 trpi) Natn. A. et al., Proc. Nat and Acad. Sci. USA, 75, 1927 (1978) J or a method analogous thereto. No power; Saccharomyces ⁇ Serebishes are preferred.
  • Saccharomyces cerevisiae AH22 has been deposited with the Fermentation Research Institute as IF 0-110 134, and is still to be deposited on September 4, 1984, with the Microorganisms Research Institute of the Agency of Industrial Science and Technology (FRI I).
  • the yeast transformant is cultured in a medium known per se, such as Burkholder's minimal medium [: Bostian, KL Proc. Natl. Acad. Sci. USA, 7, 4505 (1980: s). Culture of yeast transformants is usually 15 1 to 403 ⁇ 4, preferably 24 to 37 ° C.
  • the reaction is carried out for 0 to 96 hours, preferably 24 to 72 hours, and if necessary, ventilation and stirring can be applied.
  • the cells are collected by a known method, suspended in a buffer, and then treated with lysozyme or sonication in the case of a transformant of Escherichia coli, or with Zymolyase (in the case of a transformant of yeast).
  • the product breaks down by mechanical destruction with a glass bead or the like.
  • P31 can be extracted more advantageously by adding a surface active agent such as Triton X-100, deoxycore, or a protein denaturant such as guanidine hydrochloride.
  • Isolation of P31 from the supernatant obtained by centrifugation may be performed by a generally known method for purifying a hydrophobic protein.
  • the P31 activity of the animal can be measured, for example, by binding the sample to cellulose paper activated with bucmane and then using 125 A (available from Dynabot Inc.) Direct immunoassay method for reaction with anti-HBsAg antibody [Fujisawa, Y. et al., Nucleic Acids Res., 1_l, 3581 (1983)].
  • a DNA encoding P31 is inserted into the 3 'end of a promoter region which can function in Bacillus subtilis or animal cells, and a known DNA is used.
  • bacteria and yeast are more preferable as a host capable of producing P31.
  • the generated P31 may or may not be glycosylated.
  • H Bs A transformant containing the g DNA by production of surface antigen gene products, the force is known to growth of the transformant itself is inhibited;, P 3 according to the present invention
  • the use of DNA encoding 1 eliminates growth inhibition and increases P31 production.
  • 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 (upper row) and the corresponding amino acid sequence (lower row).
  • Fig. 3 shows the construction diagram of plasmid PPH017-1 with the symbols E, S, B,
  • H and X represent EcoRI, Sa1I, BamHI, HindI, and Xhol, respectively.
  • - Figure 4 shows the construction diagram of the plasmid pPKT 700-1 with the symbols E, 'S, ⁇
  • H and X represent EcoRI, Sa1I, BaniHI-, HindI and Xhol, respectively.
  • FIG. 5 shows a construction diagram of the plasmid PGLD906-1, wherein symbols, S, B, H and X represent EcoM, Sa1I, BamHI, Hindf and Xhol, respectively.
  • FIG. 6 shows a construction diagram of the expression plasmid pTRP P31-R for adr-type HBsAgP31 for Escherichia coli, where the symbols, B, C and JP represent EcoRI, BamHI, Clal and PstI, respectively.
  • FIG. 7 shows a construction diagram of the expression plasmid pTRP P31 -W2 for adw-type HBsAgP31 for Escherichia coli, where the symbols _E, B, C and P represent EcoRI, BamHI, Clal and PstI, respectively.
  • Figure 8 shows the expression plasmid for adr-type HBsAgP31 for yeast pGL DP31 left-handed fOMPI — R, pPHO P3 1—R and pPKT P31—R are shown in the construction diagram, symbols E, B, S, H, X and C are EcoRI, BamHI, SalI, Hindi, XhoI and C1 respectively. a Represents I.
  • FIGS. 9 and 10 show the construction diagrams of the expression plasmid pPHO P 3 ⁇ —W of the adw-type HBsHgP31 for yeast, and the symbols B, P, B, C, S and H are EcoRI and Pst I, respectively. , BamHI, C1aI, Sa1I and HindH. -Best mode for carrying out the invention
  • T4 polynucleotide 'kinase (Takara Shuzo) in 50 ⁇ £ reaction solution [5 OmM Tris-HCl (pH 7. ⁇ ), 10 mM MgCl 2 , 10 mM 5'-end was phosphorylated at 37 ° C for 1 hour in mM 2 -mercaptoethanol, 100 M ATP].
  • the reaction solution [40 mM potassium phosphate buffer (pH 7.5), 6.6 mM MgCl 2 , 1 mM 2-mercaptoethanol i 33 ⁇ d ATP, 33 M dGTP, 33 ⁇ ⁇ dTTP, 33 ⁇ dCTP] at 12 ° C for 30 min.
  • the DNA fragment was blunt-ended, deproteinized with phenol, and precipitated with cold ethanol.
  • the 294 strain was transformed according to the method of Cohen et al., And the plasmid pPHOl2 in which the Xhol site of the plasmid pPHOl2 was changed to the Sail site was selected from the ampicillin-resistant transformants. 12-1 was obtained (see Fig. 3).
  • DNA was precipitated by adding cold ethanol. After electrophoresis of DNA 0.5 ⁇ ⁇ from each fraction using 1% agarose 'slab gel under the conditions described in Reference Example 1, nitrocellulose finoleta C Schleicher and Schul 1; [Southern, EM, J. Mol. Biol “9_8, 503 (1975)]
  • TEN buffer (1 OmM Tris-HC1 (pH 8.0, 200 mM)
  • the reaction solution was used to transform E. coli DH1 CManiatis, T. et al., Molecular Cloning, Co Id Spring Harbor Laboratory, 254-255 (1982)], and about 1 transformant showing tetracycline resistance was transformed. I got 300 pieces. From among these, transformants containing the PGK gene were cloned using the above-mentioned 32 P-labeled synthetic probe in a colony 'Hive: Lydidase [Suggs, SV et al., Proc. Natl. Acad. Sci.
  • Gr..P Separated from the gel by the method described in 1.
  • 10 units of the restriction enzyme Xhol (Takara Shuzo) [30 mM reaction solution [10 mM Tris-HCl (pH 7.5, 7 mM MgCl 2 , 100 mM NaCl, 7 mM 2- mercapto).
  • electrophoresis was performed using 0.7% agarose 'slab gel under the conditions described in Reference Example 1. After electrophoresis, refer to the 7.7 kb DNA fragment. It was separated from the gel by the method described in Example 1 (see Fig. 4).
  • the plasmid pPKT567DNA10 described in 2 of Reference Example 2 was combined with 10 units of each restriction enzyme Hindi [Xhol with 0 ⁇ ⁇ reaction solution (50 mM Tris-HCl C pH 7.6), 50 mM NaCl, After incubating for 2 hours at 37 ° C in 1 mM dithiothreitol, 10 mM MgCl 2 ), electrophoresis was performed using 1.2% agarose-slab gel under the conditions described in Reference Example 1. The Okb DNA fragment was separated from the gel (see FIG. 4).
  • Escherichia coli 1 was transformed by the method described in 1 to obtain about 1200 tetracycline-resistant transformants, which were strongly hybridized with the 32 P-labeled probe by colony hybridization. Transformants were separated. Plasmid PGLD9 was isolated from this transformant by the above-described extraction method and digested with HindJI.A 2.2 kb insert DNA was detected, and the insert DNA was analyzed by jSouthern method. Hybridization was confirmed (see Fig. 5).
  • the 0.36 kb DNA fragment 1 was reacted with a DNA polymerase I large 'fragment under the conditions described in Reference Example 1 to change the cohesive end of aq I to a blunt end.
  • 1 ⁇ 9 of this fragment and 50 n of the phosphorylated Xhol linker described in Reference Example 1 were mixed, and allowed to bind by the action of T4 DNA ligase under the conditions described in Reference Example 1.
  • After the reaction add an excess amount of Xhol and allow it to work at 37 ° C for 4 hours.
  • use a Sepharose 4B column to bind the linker-bonded 0.36 kb DNA under the conditions described in 2 of Reference Example 2. The fragments were separated.
  • the DNA fragment 6 ⁇ was added with 2 units of the restriction enzyme HhaI in a 50 reaction solution [10 mM Tris-HC (pH 7.5, 50 mM NaCl, 10 mM MgCl 2 , 1 mM dithiothreitol)].
  • a 50 reaction solution 10 mM Tris-HC (pH 7.5, 50 mM NaCl, 10 mM MgCl 2 , 1 mM dithiothreitol)].
  • HhaI restriction enzyme
  • a plasmid PGLD906-1 converted to the XhoI3 ⁇ 43 ⁇ 4i force sSall site of the plasmid PGLD906 was prepared (see FIG. 5).
  • + Replacement Example 1 Construction of recombinant DNA molecule expressing adr-type hepatitis B virus surface antigen P31 gene and transformation of Escherichia coli with the DNA molecule
  • the plasmid pBR322-BamHINO HBr33ODNA (abbreviated as pHBr330) described in JP-A-59-79485 and Nucleic Acids Res., Above, 1747 (1983).
  • pHBr330 The plasmid pBR322-BamHINO HBr33ODNA (abbreviated as pHBr330) described in JP-A-59-79485 and Nucleic Acids Res., Above, 1747 (1983).
  • a reaction solution of 100 / i (100 mM Tris-) with BamHI was added to the plasmid pHBr 330 50 / ⁇ with a restriction solution of 20 units each of EcoRI (Takara Shuzo) and BamHI.
  • HC 1 pH 7.5
  • the DNA polymerase I large fragment was allowed to act on 50 ong of BamHI plasmid pHBrP31 under the conditions described in Reference Example 1 to make the adhesive end blunt, and then removed with phenol.
  • the protein was precipitated and the DNA was precipitated with cold ethanol.
  • Ligation was carried out by the action of T4 DNA ligase under the conditions described in Example 1.
  • the reaction solution was used to transform E. coli 294 strain, and the transformant was examined by the method described in Reference Example 1, and the BamHI site of the plasmid pHBrP31 was converted to Pstj;
  • the plasmid pHBrP 31-17 was separated (see Figure 6).
  • reaction solution 100 units of the pHBrP 31-1750 ⁇ with 20 units of each of the restriction enzyme Clal and Pstl (Takara Shuzo)! After reacting for 3 hours at 37 ° C in 20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 50 mM (NH 4 ) 2 SO 4 ], the reaction solution was purified using 1.0 agarose-slab gel. Electrophoresis was performed under the conditions described in Reference Example 1. After electrophoresis, 1.42 kb DNA The fragment was separated from the gel by the method described in Reference Example 1.
  • the strain (Escherichia coli 294 / pTRP P31-R) was deposited with the Fermentation Research Institute as IFO-144355, and from July 10, 1984, Deposited with the Technical Research Institute (FRI) under the accession number F ERM P—77009.
  • Example 2 Construction of recombinant DNA molecule expressing adw-type hepatitis B virus surface antigen P31 gene and Escherichia coli using the DNA molecule
  • the plasmid pBR—EcoRI / HBV933 DNA (abbreviated as pHBV933) described in Japanese Patent Application Laid-Open No. 58200/1976 and Nucleic Acids Res ,, 11, 17747 (1983) is a special feature. It was prepared by the method of Reference Example 1 described in No. 5 -2 01 796 6 ⁇ . 2 ⁇ Add 2 units of restriction enzyme Hpa to the plasmid
  • coli was The 294 strain was transformed, and the transformant was examined by the method described in Reference Example 1 to obtain a plasmid pHBV933-5 in which the Hpal site of the plasmid PHBV933 was converted to a Pstl site (No. 1). See Figure 7).
  • reaction solution 800 ⁇ ⁇ of the plasmid pHBVg 33-5 DNA and 500 units of restriction enzyme Pstl! : 20 mM Tris-HCl (pH 7.5), 1 OmM MgCl 2 , 50 mM (NH 4 ) 2 SO 4 ] at 37 ° C. for 20 minutes, and immediately deproteinized with phenol.
  • the reaction solution was subjected to electrophoresis using 1.0% agarose slab gel under the conditions described in Reference Example 1. After the electrophoresis, a 1.7 kb DNA fragment partially digested with Pstl was fractionated from the gel by the method described in Reference Example 1 (see FIG. 7).
  • the Escherichia coli strain (Esche-richia coli 294 / pTRP P31-W2 :) carrying the plasmid pTRP P31-W2 was deposited with the Fermentation Research Institute as IF0-144356. It was deposited with the Research Institute of Microbial Industry and Technology (FRI) on July 10, 1980 under the accession number F ERM P-770. ⁇
  • Example 3 Construction of a recombinant DNA molecule for yeast expressing the adr-type hepatitis B virus surface antigen P31 gene and transformation of yeast with the DNA molecule
  • the reaction solution was subjected to electrophoresis using 1.0% agarose 'slab gel under the conditions described in Reference Example 1. After the electrophoresis, a 1.42 kb DNA fragment was fractionated from the gel by the method described in Reference Example 1.
  • AH22R-1 was transformed by the method of Hinnen et al.
  • the yeast transformant (AH22R-no pPHOPa 1- R) that was retained was isolated.
  • transformants can be cultured by a usual method to obtain the desired P31.
  • Example 4 adw-type hepatitis B virus surface antigen P31 gene is expressed-construction of recombinant DNA molecule for yeast and transformation of yeast by said DNA molecule. '. 1 described in Example 2 Plasmid PHBV 933 DNA, 2 ⁇ 2.
  • Electrophoresis was performed using 1.2% agarose slab gel under the conditions described in Reference Example 1. After electrophoresis, contains DNA fragment encoding adw-type P31
  • the 0.96 kb DNA fragment was separated from the gel by the universal method described in Reference Example 1 (see FIG. 9). .
  • the DNA polymerase I large 'fragment was allowed to act on the 0.98 kb DNA fragment of 2 ⁇ under the conditions described in Reference Example 1 to make the cohesive ends blunt, followed by deproteinization with phenol and cold ethanol. DNA was precipitated. According to the method described in (3) of Example 3, the 0.98 kb DNA fragment was ligated with 5'-P-d (CGTCGACG :), Bethesda Research Co., Ltd. The fraction containing the 0.99 kb DNA fragment (DNA encoding adw-type P31) was collected using Sepharose 4B column, and the DNA was precipitated with cold ethanol (No. 9).
  • C T4D NA under the conditions described in Reference Example 1 was obtained by combining 200 ng of SalI Kouyida pPHO 17-1, 200 ng described in Example 3, and 200 ng of the above-mentioned 0.99 kb DNA fragment. It was bound by the action of ligase. Using the reaction solution, E. coli 294 strain was transformed. The transformant was prepared by the method described in Reference Example 1, and a 0.99 kb DNA fragment containing DNA encoding adw-type P31 was transformed into PHO. A strain (Escherichia coli 294 / pPHO P31-W) carrying 5 promoters and the plasmid pPHO P31-W inserted in the forward direction was isolated.
  • the yeast host AH22R-1 is transformed by the method of Hinnen et al., And the yeast transformation retaining the plasmid is performed.
  • Sacha-romyces cerevisiae AH22R ⁇ / pPHOP31-W was isolated (see Fig. 9).
  • strains Sacharomyces cerevisiae AH 22 R- / pPHO P 31- W
  • I FO- 1 0 1 3 6 the Institute for Fermentation, also, industrial technology from September 4, 1984 year Institute of Microbiological Technology (FR replacement) 1) Accession number? £ 1 ⁇ ] ⁇ P — deposited as 7 8 2 6
  • a plasmid containing DNA encoding adw-type P31 and a PGK promoter or a GLD promoter can also be constructed according to the above method.
  • the above plasmid pTRP P31-W2 was added with 20 units of the restriction enzyme Pstl to a reaction solution of 100 [10 mM Tris-HCl (pH 7.5), 10 mM ⁇ g C12, 50 mM NaCl, l After reacting at 37 ° C for 20 minutes in 37 mM dimethylthiothiol), the reaction solution is subjected to electrophoresis using 0.8% agarose-slab gel under the conditions described in Reference Example 1. . 'After electrophoresis, separate the 4.6 kb linear DNA molecule that has been cleaved at one site with Pstl from the gel by the method described in Reference Example 1 (see Fig. 10).
  • This transformant can be cultured by a usual method to obtain the desired P31.
  • Each transformant containing the P31 gene expression plasmid obtained in Examples 1 and 2 was cultured in M-9 medium containing 1.0% glucose and 1.0% casamino acid at 37 ° C for 6 hours. The cells were collected and washed with a buffer (30 mM Tris-HCl (pH 8.0), 50 mM ⁇ a C 1, 5 mM EDTA:! The cells were washed with 10 mM Tris-HCl (pH 8.0). Lysate consisting of 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, and 5 lysozyme And lysed.
  • the HBsAgl unit is the number of counts of the anti-HBsAg antibody attached to 1 ng of HBsAg small particles Auslia ⁇ —125 kit attached to 125 A.
  • Example 6 Expression of P31 gene in yeast-Example Each yeast transformant containing the P31 gene expression plasmid obtained in Examples 3 and 4 was cultured in Burkholder and its low-phosphate medium at 30 ° C for 2 days, and the cells were collected. Washed with physiological saline.
  • the recombinant D D ⁇ provided by the present invention and the transformant containing the recombinant DNA are useful for producing HBsAg P31 and provided by the present invention.
  • HBsAg P31 is useful as a vaccine useful for preventing HBV infection.

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Abstract

L'antigène P31 de surface du virus de l'hépatite B peut être préparé par culture d'un transformant contenant de l'ADN recombinant possédant de l'ADN de codage du P31 introduit dans une terminaison 3' de la région du promoteur.
PCT/JP1984/000423 1984-07-11 1984-09-04 Adn recombinant et son utilisation WO1986001534A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
PCT/JP1984/000423 WO1986001534A1 (fr) 1984-09-04 1984-09-04 Adn recombinant et son utilisation
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
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

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Citations (3)

* Cited by examiner, † Cited by third party
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
JPS5980615A (ja) * 1982-10-29 1984-05-10 Takeda Chem Ind Ltd Ｄｎａおよびその用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
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
JPS5980615A (ja) * 1982-10-29 1984-05-10 Takeda Chem Ind Ltd Ｄｎａおよびその用途

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