EP0105149A2
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EP
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Recombinant plasmid containing hepatitis B virus gene, yeast transformed with said recombinant plasmid, and production of hepatitis B virus surface antigen
Assignee: Science and Technology Agency Minister's Secretariat Director of Finance Division
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Filing: 1983-08-16
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Granted: —
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Expires: 2003-08-16
Abstract
Abstract Described are recombinant plasmids containing a Hepatitis B virus gene, which comprises a plasmid vector containing a yeast derived DNA and a E. coli derived DNA and carrying the expression control region of the repressible acid phosphatase gene of yeast and a Hepatitis B virus gene recombined thereto under control of the phosphatase promoter, a transformed yeast which is prepared by transforming a yeast with the recombinant plasmid, and a method of the production of Hepatitis B virus surface antigen in a large scale by cultivating the transformed yeast in a medium. The Hepatitis B virus surface antigen prepared by the present invention has the same immunological properties as those of the natural antigen from hunan blood plasma and is useful for the preparation of Hepatitis B virus vaccine and diagnostic reagents.
Inventors
- 👤 Atsushi Miyanohara
- 👤 Chikateru Nozaki
- 👤 Fukusaburo Hamada
- 👤 Akio Toh-E
- 👤 Nobuya Ohtomo
- 👤 Kenichi Matsubara
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-09 23:18
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/005; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/80; C12N15/81; C07K2319/00; C07K2319/40; C12N2730/00; C12N2730/00011; C12N2730/10011; C12N2730/10111; C12N2730/10122
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
49 words
A recombinant plasmid, which comprises a plasmid vector containing a yeast derived DNA and a Escherichia coli derived D N A and carrying the expression control region of the repressible acid phosphatase gene of yeast and a Hepatitis B virus gene recombined thereto under control of the phosphatase promoter.
Claim 2
Dependent Claim
49 words
A recombinant plasmid, which comprises a plasmid vector containing a yeast derived DNA and a Escherichia coli derived D N A and carrying the expression control region of the repressible acid phosphatase gene of yeast and a Hepatitis B virus gene recombined thereto under control of the phosphatase promoter.
Claim 3
Dependent Claim
59 words
The recombinant plasmid according to claim 1, wherein said expression control region of the repressible acid phosphatase gene of yeast is a gene coding for a polypeptide of 60',000 daltons (p60) which constitutes the acid phosphatase, a part or the complete structural gene of the acid phosphatase and optionally a certain region upstream the phosphatase structural gene being deleted.
Claim 4
Dependent Claim
37 words
The recombinant plasmid according to claim 2, wherein the region to be deleted upstream the phosphatase structural gene in addition to the complete phosphatase structural gene is in the range of from +1 (ATG) to -100 bp.
Claim 5
Dependent Claim
23 words
The recombinant plasmid according to claim 3, wherein the region to be deleted is in the range of from +1 to -50 bp.
Claim 6
Dependent Claim
19 words
The recombinant plasmid according to claim 1, wherein said yeast derived DNA includes ars 1 and 2 µ ori.
Claim 7
Dependent Claim
27 words
The recombinant plasmid according to claim 1, wherein said yeast derived DNA includes ars 1, 2 µ ori and a selective marker gene for a transformed yeast.
Claim 8
Dependent Claim
45 words
The recombinant plasmid according to claim 6, wherein said selective marker gene is a member selected from the group consisting of a leucine-producing gene, a histidine-producing gene, a tryptophane-producing gene, a uracil-producing gene, and an adenine-producing gene, and a combination of two or more thereof.
Claim 9
Dependent Claim
28 words
The recombinant plasmid according to claim 1, wherein said E. coli derived DNA includes a DNA sequence which is necessary for replication of the DNA in E. coli.
Claim 10
Dependent Claim
38 words
The recombinant plamid according to claim 1, wherein said E. coli derived DNA includes a DNA sequence which is necessary for replication of the DNA in E. coli. and a selective marker gene for a transformed E. coli.
Claim 11
Dependent Claim
39 words
The recombinant plasmid according to claim 9, wherein the selective marker gene is a member selected from the group consisting of an ampicillin-resistance gene,.a kanamycin-resistancegepe, a tetracycline-resistance gene and a chloramphenicol-resistancegene, and a combination of two or more thereof.
Claim 12
Dependent Claim
25 words
The recombinant plasmid according to claim 9, wherein said E. coli derived DNA is the plasmid pBR322 containing an ampicillin-resistance gene and a tetracycline-resistance gene.
Claim 13
Dependent Claim
87 words
The recombinant plasmid according to claim 1, wherein said plasmid vector is a shuttle vector containing a yeast plasmid and a E. coli plasmid, said yeast plasmid containing ars 1, 2 µ ori, leu 2 and the expression control region of the repressible acid phosphatase gene in this order wherein a part or the complete acid phosphatase structural gene and a certain region upstream the phosphatase structural gene are deleted, and said E. coli plasmid being . the plasmid pBR322 which contains an ampicillin-resistancegene and a tetracycline-resistancegene.
Claim 14
Dependent Claim
37 words
The recombinant plasmid according to claim 12, wherein said yeast plasmid is deficient of the complete acid phosphatase structural gene and of a. region upstream the phosphatase structural gene in the range of +1 to -100 bp.
Claim 15
Dependent Claim
26 words
The recombinant plasmid according to claim 13, wherein the deficient region upstream the phosphatase structural gene is in the range of from +1 to -50 bp.
Claim 16
Dependent Claim
34 words
The recombinant plasmid according to claim 1, wherein said Heptatits B virus gene to be recombined with the plasmid vector is a fragment containing the complete H Bs gene corresponding to 226 amino acids.
Claim 17
Dependent Claim
30 words
The recombinant plasmid according to claim 15, wherein said fragment containing the complete H Bs gene is the complete HBV DNA which is cleaved with a restriction enzyme Xho I.
Claim 18
Dependent Claim
31 words
The recombinant plasmid according to claim 15, wherein said fragment containing the complete HBs gene is a fragment prepared by cleaving the HBV DNA with restriction enzymes Xho I and BamHI.
Claim 19
Dependent Claim
20 words
A transformed yeast which is prepared by transforming a yeast with the recombinant plasmid as set forth in claim 1.
Claim 20
Dependent Claim
41 words
The transformed yeast according to claim 18, wherein said starting yeast is a member selected from the group consisting of a leucine-requiring mutant strain, a histidine-requiring mutant strain, a tryptophane-requiring mutant strain, a uracil-requiring mutant strain, and an adenine-requiring mutant strain.
Claim 21
Dependent Claim
27 words
The transformed yeast according to claim 18, wherein the starting yeast is a leucine-requiring mutant strain: Saccharomyces cerevisiae AH22 [a, leu2, his4, Can 1 (Cir + )].
Claim 22
Dependent Claim
34 words
A method for production of Hepatitis B virus surface antigen, which comprises cultivating the transformed yeast as set forth in claim 18 in a medium and collecting the produced antigen by a conventional method.
Claim 23
Dependent Claim
27 words
The method according to claim 21, wherein the cultivation of the transformed yeast is carried out under the conditions that an acid phosphatase promoter is not repressed.
References Cited (Backward Citations)
No citation records indexed.
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