WO1986005808A1 - Nouvel adn et son utilisation - Google Patents

Nouvel adn et son utilisation Download PDF

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Publication number
WO1986005808A1
WO1986005808A1 PCT/JP1985/000161 JP8500161W WO8605808A1 WO 1986005808 A1 WO1986005808 A1 WO 1986005808A1 JP 8500161 W JP8500161 W JP 8500161W WO 8605808 A1 WO8605808 A1 WO 8605808A1
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Prior art keywords
hbsag
dna
plasmid
hepatitis
surface antigen
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PCT/JP1985/000161
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English (en)
Japanese (ja)
Inventor
Masakazu Kikuchi
Kazuo Nakahama
Yukio Fujisawa
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Takeda Chemical Industries, Ltd.
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Publication date
Application filed by Takeda Chemical Industries, Ltd. filed Critical Takeda Chemical Industries, Ltd.
Priority to PCT/JP1985/000161 priority Critical patent/WO1986005808A1/fr
Priority to JP409286A priority patent/JPS61231997A/ja
Publication of WO1986005808A1 publication Critical patent/WO1986005808A1/fr

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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 DMA and its use.
  • DNA at the 3 'end of D ⁇ which codes for the hepatitis B virus surface antigen is deleted, and a promoter region is located upstream of the DNA and a stop codon is located downstream.
  • the present invention relates to a novel protein having a surface antigen effect and a method for producing the protein.
  • Hepatitis B is a viral disease that occurs frequently in tropical Africa, Southeast Asia and the Far East, and has been epidemiologically suggested to cause chronic hepatitis, cirrhosis, and even primary liver cancer. I have.
  • the etiology is the hepatitis B virus (HBV), a member of the NA virus, which bears the name of the discoverer with a 42-nm spherical particle. They are called Dane particles.
  • HBV surface antigen hereinafter abbreviated as HBsAg
  • HBsAg HBV surface antigen
  • P25 and P31 are mainly known as HBsAg depending on the molecular weight, and P31 is obtained by adding 55 amino acids to the N-terminal of P25.
  • HBsAg In addition to den particles, small particles and tubular particles were detected in the blood of hepatitis B patients, and the same type of HBsAg as den particles was found in these particles. Antibodies against viral superficial antigens protect against viral infection This is also known for other viruses. In the case of HBV, a vaccine against hepatitis B may be produced based on HBsAg. However, HBV only infects 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 particles and the like only satisfy the demand as materials for diagnostic reagents, making it impossible to use them for vaccine production. State.
  • HBsAg HBsAg structural gene
  • HBsAgP25 has been reported to be expressed in Escherichia coli as a fibrillary protein. [Charnay, P. et al., Nature, 286, 893 (1980); Edman, J. C. et al., Nature, 291, 503 (11981)].
  • HBsAg P 2 5 high producing strain HBs Ag F 2 5 result of changing process one line producing, 1 0 one 5 frequency of H BsAg F 2 5 production High strains were successfully isolated.
  • the C-terminal side of HBsAgP25 was deleted due to the insertion sequence IS1 (Insertional Sequence 1) on the HBsAgP25 gene.
  • HBsAgP25 was produced in large quantities.
  • an adapter having a stop codon was chemically synthesized and inserted into a restriction enzyme cleavage site in the HBsAg P25 gene, whereby a large amount of HBsAg P25 with a C-terminal deletion was produced.
  • the present invention was completed by knowing that this knowledge can be applied to improving the production of HBsAg P31.
  • the present invention is a.
  • the DNA coding for the hepatitis B virus surface antigen lacks a portion of the DNA at the 3 'end, and has a promoter region upstream and a stop codon downstream, and (2) the hepatitis B virus surface.
  • a transformant that lacks a portion of the 3'-terminal DNA of the DNA encoding the antigen contains a promoter region upstream of the DNA, and a DNA having a stop codon downstream, 3 hepatitis B Culturing a transformant containing a promoter region upstream of the 3'-end of DNA encoding the virus surface antigen, a promoter region upstream thereof, and a stop codon downstream thereof
  • a novel protein having hepatitis B virus surface antigen activity of producing a novel protein having hepatitis B virus surface antigen activity
  • the present invention relates to a novel protein having a hepatitis V virus surface antigen activity, which lacks a part of the C-terminal side of the hepatitis V virus surface antigen and optionally has an amino acid or a peptide different from that in the defective portion.
  • the DNA encoding HBsAgP25 used in the present invention in which a portion of the DNA at the 3′-terminal side of the DNA is deleted can be prepared, for example, by the following method.
  • E. coli C600 / pTRPSS-6 described in the literature [eg, ucleic Acid Research, 11, 3581 (19983)] can be used as a drug, eg, N-methyl- N '- by mutation treatment with nitro one N- Nitorosogua two gin, it is possible to separate the good strains growth on plates 1 0 one second frequency at.
  • HBsAgP can also be introduced by introducing pTRP SS-6 or an adapter having a stop codon at the restriction enzyme cleavage site on the HBsAgP25 gene of plasmid in which insertion of IS1 has been recognized. It is possible to obtain a DNA in which a part of the DNA at the 3 'end of the DNA encoding 25 is deleted.
  • Subtypes other than adw-type HBsAg P25 such as adr-type, ayr-type, and ayw-type HBsAg-P25-specific DNAs that lack some of the DNA at the 3 'end of the DMA It can be easily obtained according to the above method based on a transformant having a DNA coding type.
  • DNA that lacks a portion of the DNA at the 3 'end of NA that encodes HBsAg P31 contains DNA that contains DXA that encodes HBsAg P31.
  • a restriction enzyme such as BstNI.
  • the deficient portion at the C-terminal side of HBsAg may have any size as long as the remaining peptide has an action as HBsAg, and the number of amino acids is preferably ⁇ 4 or more and 999 or less.
  • HBsAg may be partially deleted at the N′-terminal side, and DNA encoding HBsAg with partially deleted N-terminal side is disclosed in, for example, One described in Japanese Patent Application Laid-Open No. 8-21076. Can be obtained by a method or a method analogous thereto. .
  • the transformant used to obtain the plasmid into which the above-mentioned IS1 was inserted was Escherichia coli having HBsAg expression plasmid, for example, Escherichia coli such as C600, JA21.2.294, etc.
  • Escherichia coli such as C600, JA21.2.294, etc.
  • Examples of the HBsAg expression plasmid used include a plasmid that expresses the HBsAg gene in Escherichia coli and produces HBsAg, such as pT RPSS-6, ⁇ R ⁇ 31-R.
  • the transformant is subjected to mutation treatment, for example, N-methylation. It may be treated with a mutagen such as N-2-nitrosoguanidine or UV irradiation.
  • mutation treatment for example, N-methylation. It may be treated with a mutagen such as N-2-nitrosoguanidine or UV irradiation.
  • a medium for obtaining a well-growing strain any medium can be used as long as the transformant can grow, but a medium having high HBsAg productivity, for example, an improved M-9 agar medium [Nucleic Acid Research, 1 1, 35881 (19983)] is preferred.
  • any method capable of easily and accurately measuring the amount of HBsAg produced may be used. , 3 5 8 1 (1 9 8 3)] Good. Isolate the plasmid from the resulting HBsAg high-producing strain, examine the HBsAg gene and its restriction enzyme cleavage map by, for example, agarose gel electrophoresis or acrylamide gel electrophoresis, and determine the base sequence. As a result, it is possible to know that an insertion sequence has been inserted into the HBs Ag gene.
  • HBsAg expression plasmid in which a part of the C-terminus has been deleted from a plasmid containing the above-mentioned insertion sequence or pTRP SS-6, ⁇ RP-31-R, etc., these plasmids must be used.
  • An adapter having a stop codon may be inserted into the restriction enzyme cleavage site on the HBsAg gene of the plasmid, for example, the Hpall site of pTRPG7 shown in Example 1 described later, and the like.
  • the adapter having a termination codon may be any adapter as long as it terminates the protein coded from the upstream of the site into which it is inserted with a termination codon, and the termination codon is ⁇ ⁇ , ⁇ GA, Any of TAG may be used. It can be easily obtained by chemically synthesizing these adapters using, for example, the triester method [Proc. Natl. Acad. Sci. USA, l_5_, 5765 (19778)].
  • the DNA lacking a part of the 3 ′ end of the DNA encoding HBsAg is added to the 3 ′ end of the promoter region that functions in various hosts (eg, Escherichia coli, Escherichia coli, yeast, animal cells).
  • hosts eg, Escherichia coli, Escherichia coli, yeast, animal cells.
  • a recombinant DNA capable of expressing a DNA in which a part of the DNA encoding HBsAg is partially deleted at the 3 ′ end can be constructed.
  • the promoter region may be any region containing a site necessary for initiating mRNA synthesis by binding of RNA polymerase.
  • HBsAg is coded.
  • a DNA that is partially deleted at the 3 'end of A is a promoter that can function in E. coli.
  • Replacement HBsAg is inserted into the 3 'end of the protein region
  • Recombinant DNA capable of expressing DNA in which a part of the 3 ′ end is deleted can be constructed.
  • E. coli eg, C600 strain, 294 strain, W3110 strain, RR1 strain, PR13 strain, etc.
  • was prepared by a known method [Cohen, S.N. proc. Natl. Acad. S ci . US a t 6 9, 2 1 1 0 (1 9 7 2)] If Ku is transformed by methods analogous thereto.
  • the promoter to be used need not be limited to the trp promoter (trp-P).
  • trp-P trp promoter
  • a recA promoter Japanese Patent Laid-Open No. 59-65099
  • a lac promoter a lac promoter
  • a PL promoter One or the like may be used.
  • a transformant harboring a novel recombinant plasmid ⁇ ⁇ containing E! NA in which the HBsAg-encoding DNA obtained as described above is partially omitted at the 3 ′ end is, for example, ampicillin-resistant.
  • Tetracycline resistance or resistance to both herbicides can be selected as the phenotype.
  • DNA encoding another amino acid or peptide may be inserted, and the HBsAg gene into which the inserted sequence is inserted may be inserted.
  • a protein having an amino acid and a peptide different from the HBsAg sequence at the C-terminal deletion can also be produced using the protein.
  • the transformant thus selected is cultured in a medium known per se.
  • Examples of the medium include an M-9 medium containing L broth, Pe'nassay broth, dalcose, and casamino acid [Miller, J. Experiments in Molecular Genetics, 431-1433 (Cold Spring Harbor Laboratory, New York, 1972).
  • a drug such as 3 ⁇ 3-indolylacrylic acid can be added to make the promoter work efficiently.
  • Culture of the transformant is usually performed at 15 to 43 ° C, preferably 28 to 40 ° C, for 2 to 24 hours, preferably 4 to 16 hours, and if necessary, aeration and stirring may be applied. it can.
  • a yeast transformant can be prepared as follows. E. coli-yeast shuttle vector YE p 13 [Broach, J., R. et al., Gene, ___, 121 (1 7979)], pSH 15 and pS-H 19 [Harashima, S. et al., Mol. Cell. Biol,, 7 7.1
  • yeast promoter region such as the inhibitory acid phosphatase gene promoter region [Meyhack. B. et al., ⁇ 0 J., 6,
  • Transformants carrying the novel recombinant DMA containing DNA encoding HBsAg with a partially deleted C-terminal side, obtained in this way, have, for example, a phenotype of ampicillin resistance. As you can choose. In this resistant strain
  • Plasmid DNA was extracted from the transformants selected in this manner by the force extraction method [Birnboim, H.C. and Doly, J., ucleic Acids Res., 7_, 1 ⁇ 13 (19) 7 9)], and use it for yeast, for example, Saccharomyces cerevisiae requiring auxin isin, for example, AH 2 2 R (leu2 his 4 can 1 cir + pho 80) [Proc. atl. Acad. Sci. US A80, 1 (1983)] or K33-7B (pho80-AH22, pho8-2) or K33 derived from AH22R- -8 D (pho 80-AH 22, pho 8-2 trpl) was converted to a known method [Hinnen, A. et al., Proc. Natl. Acad. Sci. US A, 75, 1972 (1 9 7 8)] or an equivalent method.
  • the yeast used as a host is not limited to these, but is preferably S. cerevisiae.
  • the obtained yeast transformant is cultured in a medium known per se.
  • the medium include Burkholder's minimum medium [Bostian, K. L. et al., Proc. Natl. Acad. Sci. US A, 77, 455 (1980)].
  • Culture of the yeast transformant is usually carried out at 15 to 40, preferably 24 to 37 ° C for 10 to 96 hours, preferably 24 to 72 hours. Can also.
  • the cells After culturing, the cells are collected by a known method, suspended in a buffer solution, and then subjected to lysozyme or ultrasonic treatment in the case of Escherichia coli transformants, or Zymoliet in the case of yeast transformants. —The cells are destroyed by mechanical destruction by glass beads or Kirin Brewery Co., Ltd.
  • surfactants such as Triton X-100, doxycholate, or salts
  • a protein denaturant such as guanidine acid allows the extraction of HBsAg with a partial deletion at the c-terminus more advantageously.
  • c-terminus from the supernatant obtained by c- centrifugal separation Isolation of HBsAg in which a portion of the HBsAg is deleted may be performed according to a generally known protein purification method.
  • the cells are collected by a known method, and in the case of a transformant of Escherichia coli, the cells are suspended in a buffer containing a protein denaturing agent such as urea or guanidine hydrochloride. After stirring, centrifugation is used to obtain a supernatant containing HBsAg with a partial deletion at the C-terminal side, or the cells are suspended in a buffer, sonicated, lysozyme and / or freeze-thawed to separate the cells. After rupture, a method of obtaining a supernatant containing HBsAg in which a portion of the C-terminal side is deleted by eccentric separation may be used as appropriate. After homogenization and lysis, a method comprising adding a buffer containing urea (3 to 10 M), stirring (0.5 to 8 hours at 0 to 10), and centrifuging to obtain a supernatant is preferred.
  • a buffer containing urea 3 to 10 M
  • stirring 0.5 to 8
  • Separation and purification of the HBsAg P31 protein from which the C-terminal portion is deleted from the above-mentioned extract is carried out by a purification process including affinity chromatography.
  • Examples include affinity chromatography using (poly-HSA) as a ligand, and antibody column treatment using an antibody against HBsAg, particularly a monoclonal antibody.
  • Affinity chromatography using poly-HSA as a ligand is extremely advantageously used for the purification of P31 protein.
  • formyl cell ⁇ -fine Seikagaku Corporation
  • Affigel-15 Bio-Rad Co., Ltd.
  • formyl cell mouth fine is particularly preferred.
  • Replacement i poly-HSA can be produced by polymerizing human serum albumin with a cross-linking agent (eg, glutaraldehyde). This to the carrier, eg if a reducing agent (such as NaCNBH 3) and combined using, to obtain a ⁇ of optionally washed responsible body and poly- HS A, use stuffed this is usually the column ⁇ You.
  • a cross-linking agent eg, glutaraldehyde
  • a P31-containing solution (cell extract supernatant) in which a part of the C-terminal side was deleted was added to a buffer solution. Adsorb to the above column equilibrated with [phosphate buffer] and elute with buffer. An appropriate amount of a surfactant (Tween20, etc.) or a protein denaturant (urea) can be added to the buffer and used as an appropriate eluate by combining the types and concentrations of these additives. .
  • the above concentrated solution is desirably reduced using a SH reagent such as dithiothreitol, and then chromatographed using a high-performance liquid chromatograph using a reversed-phase column or a hydrophobic column such as hydrophobic chromatography. Preferably, it is subjected to one treatment.
  • a SH reagent such as dithiothreitol
  • alkyl (C -! 18 about 0) of gay-containing ones e.g., AP- 2 0 2 3 0 0 A (C 8) (YMC ShimaHisa), Ultra pore RP SC (base Dzukuman Co.), Hipore RP 3 04 (Bi Ora' de Co.) and the like, especially AP- 2 0 2 3 0 0 A (C 8) are preferably used.
  • an alkylated (about d- 18 ) carrier for example, butyl toyopearl (Toyo Soda Kogyo): Octyl Sepharose (Pharmacia) and the like can be mentioned.
  • an alkylated (about d- 18 ) carrier for example, butyl toyopearl (Toyo Soda Kogyo): Octyl Sepharose (Pharmacia) and the like can be mentioned.
  • the obtained fraction containing the P31 protein partially deleted at the C-terminal side can be subjected to lyophilization, if desired, to give a white powder.
  • the sample is reacted with a plastic plate coated with poly-HSA. Thereafter, the P31 protein with a partial deletion at the C-terminal end bound to poly-HSA was detected using an anti-HBsAg monoclonal antibody conjugated to horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • the F31 protein with a partial deletion at the C-terminal end bound to poly-HSA was synthesized by ELISA (Analytica, Dynabot, USA). ) of 125 I - it can be used radios immuno mediation Si (RIA) technique that detected with anti-HBsAg antibody.
  • the HBsAg protein partially deleted at the C-terminal side produced by the method of the present invention can be used for immunological analysis similar to that of a known HBsAg small abductor produced from blood of a known HBV infected person. It has biological properties and can be used as a vaccine for diagnosis, prevention and treatment of HBV infection in the same manner as the HBsAg small particles.
  • the HBsAg activity in which a part of the C-terminal side of the product is deleted can be determined, for example, by binding the sample to cell broth-activated paper and then using the 125 I-anti-HBsAg monoclonal antibody described above.
  • the reaction is performed by direct imm, noassay method, F uj isawa, Y. 3 ⁇ 4>, ucleic Acids Res., 1 1, 3 5 8 1 (1 9 8 3)].
  • HBsAg-encoding DNA at the 3 'end of the promoter region capable of functioning in Bacillus subtilis or animal cells is partially deleted at the 3' end.
  • HBsAg By introducing DNA, transforming a host with the recombinant DNA by a method known per se, and culturing the transformant, HBsAg with a partial deletion at the C-terminal side can be produced.
  • Escherichia coli and yeast are more preferred as hosts.
  • the resulting HBsAg lacking a part of the C-terminal side may be glycosylated or non-glycosylated.
  • a transformant containing HBsAg DNA inhibits the growth of the transformant itself by production of a surface antigen gene product. 'Using a DNA with a partial deletion at the terminal end releases growth inhibition and increases the production of HBsAg with a partial deletion at the C-terminal side.
  • FIG. 1 shows plasmid PT RP 07 ⁇ 1 ⁇ 11? The construction diagram of G71 is shown.
  • Figure 2 shows a restriction map of the HBsAg gene of plasmids pTRP SS-6 and pTRP G7, where Hp is Hpal, X is Xbal, S is Sau3 AI, B is BstNl, A indicates Avail, Ha indicates Hpall, and P indicates Fst I.
  • FIG. 3 shows the nucleotide sequence of the region encoding the amino acids at positions 120 to 130 of HBsAg of plasmids pTRP SS-6, ⁇ RPG7, ⁇ RPG71 and PTRPG12. 1 shows an amino acid sequence.
  • FIG. 4 shows the construction diagram of plasmid pTRPG12.
  • FIG. 5 shows a construction diagram of plasmid pTRP P31-R
  • FIG. 6 shows a construction diagram of plasmids pT, RP. P31-212 and pTRF P31-227.
  • FIG. 7 shows the nucleotide sequence of DNA encoding adr-type HBsAgP31.
  • HBsAg small particles 50 "g obtained from human serum as an antigen were mixed well with Freund's complete adjuvant in 6-8 / 8 female mice aged 7-8 weeks, and then intraperitoneally. Two weeks after the first immunization, the same amount of antigen was mixed well with Freund's complete adjuvant, and inoculated intraperitoneally (secondary immunization). A third immunization was performed in the same manner as the second immunization.6 days after the second immunization, a part of the blood was collected from the mice, and the antibody titer in the serum was measured by the EIA method. Dissolve 100 / zg of antigen in 0.5 ml saline solution
  • the final immunization was carried out by inoculating the immunized substance intravenously. Three days after the final immunization, the spleen was excised from the NO. 2 mouse, compressed and filtered with a stainless steel mesh, and suspended in Fig. Minimal Minimum Essential Medium (MEM) to obtain a spleen cell suspension. .
  • BAL BZC mouse myeloma cells P3—X63.Ag8. ⁇ 1 ( ⁇ 3U1) were used as the cells for cell fusion (Current Topics in Microbiology and Immunology, Vol. 81, p. 1) , 1978). The cell fusion was carried out according to the original method (Neichii, 256, 495, 1979).
  • the spleen cells and P3U1 were each washed three times with serum-free MEM, and the spleen cells and P3U1 were mixed at a ratio of 5: 1 to 5: 1.
  • the cells were pelleted by centrifugation for minutes. After sufficient removal of the supernatant, the pellet was loosened gently, and 45% polyethylene daricol (PEG) 600-(manufactured by Kochlite) (3 ml) was added. After 2 minutes, add MEM to the cells at a rate of 2 ml per minute, add a total of 12 ml of MEM, and centrifuge at 600 rpm for 15 minutes to remove the supernatant.
  • PEG polyethylene daricol
  • RPM I 1 64 0 medium RPM I 1 64 0 - 1 OFCS
  • RPM I 1 64 0 - 1 OFCS cell ⁇ product RPM I 1 64 0 medium
  • inoculated in a 24-well multi-dish manufactured by Rempro
  • the cells were cultured at 37 ° C in a 5% CO 2 gas flannel.
  • PRMI 164 0-10 FCS medium HAT (hypoxanthine 1 X 10 _ + M.
  • HAT selective culture was started HAT selective culture was continued 3.5 to 7 days after the start of culture, by discarding 1 ml of the old solution and adding 1 ml of HAT medium. after one observed in the 0-1 four days, when the culture broth turned yellow (about 1 X 1 0 6 / ml) , and the supernatant
  • SS-50 was inoculated on a Millipore HA filter (manufactured by Millipore Corporation) placed on a Brain Heart Infusion (manufactured by Difco) agar medium, and incubated at 37 ° C overnight overnight.
  • the above filter was transferred to an M'-9 plate to which 40 g / ml of 3 indolylacrylic acid was added, and 37. (: 6 hours.)
  • the colonies grown on the filters were then combined with the colony-iminoase method reported by Clarke et al. [Meth.
  • ⁇ i One-well (S-06) hybridomas were cloned by limiting dilution. That is, two hybridomas were suspended in RPMI 164-20 FCS so as to be 2 ml Zml, and 0.1 ml per i-well was dispensed into a 96-well microplate (manufactured by Nunc). At the time of dispensing, thymocytes of a BAL B / C mouse were added as feeder cells at 5 ⁇ 10 5 cells / well. In this way, cell proliferation began to be observed about 2 weeks later. The supernatant was collected and examined for the presence of antibodies by the RIA method. Antibody activity was observed.
  • 2 0 black is one of Ichin S- 0 6- 04 clonal cell 1 X 1 0 6 cells are seeded into the peritoneal cavity of BAL B ZC mice that had been administered in advance 0.5ml of mineral oil intraperitoneally As a result, he became angry. High Priestess de '- Ma and after 1 0 days of administration to the abdominal cavity, ascites was collected at the antibody titer was 5 a measurement at the EIA method, it showed antibody activity up to 1 0 7 dilution. Incidentally, the antibody activity in the culture supernatant of the clone cells, mosquitoes as permitted until 1 0 4 fold dilution, about 1 0 0 0 times antibody activity by abdominal Korika rose.
  • Monoclonal antibody was purified according to the method of (1989). First 0 1 for removing Fipuri down-like substance from the ascites, 0 0 0 After centrifugation rpm ⁇ 5 minutes, re-phosphate buffer - saline (PBS: 8.1mM-NaH 2 P 0 4 .1.5mM KH 2 P 0 +, 2.7mM KC 1, 1 3 7 mM NaC and D ultraviolet absorption of 2 8 0 nm at pH 7.2) (a 28.) was Mare ⁇ the concentration shown the value of 1 2 to 1 4. After dilution, add a saturated ammonium sulfate solution to the sample to a concentration of 47%.
  • PBS 8.1mM-NaH 2 P 0 4 .1.5mM KH 2 P 0 +, 2.7mM KC 1, 1 3 7 mM NaC and D ultraviolet absorption of 2 8 0 nm at pH 7.2
  • the mixture was centrifuged for 60 minutes with stirring at 4, and then centrifuged (10, 00 rpm, 15 minutes) to obtain a sediment.
  • the precipitate was dissolved in 20 mM Tris buffer solution (PH7.9) containing 50 mi NaCl, and the solution 2 was dialyzed. Two hours later, 22 new same Tonyi liquids were used and another 15 hours of Tonyxin. Toru ⁇ , precipitation performed 1 0.0 0 O rpml 5 minutes centrifugation to remove the supernatant was adjusted to the value of the A 280 to a concentration of 2 0-3 0.
  • the sample was applied to an 8 ml DE AE cell-mouthed source column (Etman DE 52 ) equilibrated with a sufficient amount of 20 mM Tris buffer solution (pH 7.9) containing 5 OmM-NaCl. Fractionation was performed at an outflow rate of ml. Under these conditions, antibody activity was mainly observed in the flow-through fraction.
  • the anti-HBsAg monoclonal antibody fraction obtained in Reference Example 3 was concentrated and adjusted to a concentration of 4 mgZml. Chloramine T method [L inde, S. et al .; Meth. Enzymol., 92, 309-335] (19983) using Na 125 I of this sample at 200 zg & 1 mCi. ].
  • E. coii C 600 / pT RPSS-6 which produces HBsAg F 25, grows poorly and its production is not sufficient yet [Fijisawa et al., Ucleic Acids Research, 11, 3 5 8 1 ( 198 3)]. Therefore, an attempt was made to obtain a well-growing strain by mutating this transformant.
  • E. coli C600 / pTRPSS-6 was cultured with shaking at 28 ° C. for 16 hours in a test tube containing 5 ml of Brain Heart Infusion (BHI) medium (Difco). Next, 0.05 nd of this culture solution was inoculated into a test tube containing 5 ml of BHI medium, and shaking culture was performed at 37 ° C for 2 hours. The cells collected by eccentric separation from the resulting culture were washed with 50 mM Tris-maleate buffer (PH 6.0),
  • the suspension was suspended in 5 ml of the same buffer. To this suspension, add N-methyl-N'-nitro-N-nitrosguanidine to a final concentration of 100 gZml, shake at 37 ° C for 30 minutes, and then use the above buffer. Washed twice and suspended in 5 ml of the same buffer. The obtained suspension was spread on a BHI agar plate containing 12.5 zg / ml of tetracycline each in G. iml, and the plate was kept at 37 ° C. overnight. The killing rate of the colonies that grew by this treatment was about 30%.
  • the obtained colonies (300,000 strains) were transformed with an improved M-9 agar plate containing tetracycline (1,5 zgZml) [Fu sawa et al., Nucleic Acid Research, 11, 358 1 (1 983), and cultured overnight at 37 to obtain a strain with a large colony, that is, a well-growing strain (300,000 strain).
  • strains (3 strains) which were considered to have a high production amount of HBsAg were also isolated by the above-mentioned direct Imuno Atssei.
  • the HBsAg of the bacterial cell extract obtained by liquid culture was determined for the purpose of investigating the amount of HBsAg produced by these three strains. That is, the strain was cultured with shaking at 37 ° C. for 6 hours in a 200 ml Erlenmeyer flask containing 20 ml of BHI medium containing tetracycline (12.5 / zg / ml).
  • One strain was selected from the above three strains that produced a large amount of HBsAg, and the plasmid was isolated. When this plasmid was used to transform E. coli C600, a strain with high HBsAg production was obtained again.
  • this plasmid was named pTRP G7.
  • this plasmid was examined by agarose gel electrophoresis, polyacrylamide gel electrophoresis, and nucleotide sequence determination.
  • the part coding for proline 127 of the HBsAg gene was bp import system ij IS 1 (E. Ohtsubo et al., Genetic Engineering, Vol. 3, J. K. S et low & A. Hollander eds, Plenum Publishing Corporation, ew York, 1981, p33 :) is inserted, so that the amino acids after the 128th amino acid are different from the natural type, and the amino acid at the 133th amino acid is the C-terminal (Figs. 1, 2, and 3).
  • Plasmid pTRPG7 (1 / zg) was cut with 5 ': / restriction enzyme PstI, and a small PstI fragment (1.06 Kb) was removed by agarose gel electrophoresis. After ligating with ligase, E. coli C600 was transformed 5 times to obtain plasmid pTRPG71 (FIG. 1) from which the 1.06 Kb PstI fragment of pTRP G7 had been removed. .
  • E. coli C600 / pTRPG71 produced HBsAg (1.2 mg / i2 of culture solution) at the same level as E. coli C600 / pTRPG7.
  • TRPG7 the introduction of IS1 creates an Hpall site in the portion coding for the 127th proline of HBsAg (see FIG. 3). Therefore, using this HpaK site, an expression plasmid for HBsAg having the 127th proline as the C-terminus was constructed as follows. -5 pTRP G7 (844 was completely digested with restriction enzymes Xbal and PstI.
  • the XbaI-PstI fragment (50 ⁇ g) obtained as described above was completely digested with the restriction enzyme Hpafl, and then subjected to 5% polyacrylamide gel electrophoresis. Xbal-HpaK fragment (3.35 zg) was obtained.
  • the plasmid pTRP SS-6 (529 g) was completely digested with restriction enzymes Xbal and PstI, and a 3.37 Kb XbaI-PstI fragment (24 1 rg).
  • the 300 XbaI-PstI fragment (1.5 g) obtained as described above and the ⁇ & I-PstI fragment (2 zg) of 3.371: 1) were ligated with T4 DNA ligase.
  • E. coli C600 was transformed.
  • Plasmid was extracted from the tetracycline-resistant transformant and named pTRPG12 (Fig. 4). This plasmid encodes HBsAg with the 127th proline at the C-terminus (see Fig. 3).
  • plasmid pTRPSS-6 was completely digested with restriction enzymes XbaI and PstI, and an XbaI-PstI fragment of 3.37 Kb was obtained by agarose gel electrophoresis and electrophoretic elution.
  • the 3.37 Kb XbaI-PstI fragment and the 380 bp XbaI-PstI fragment obtained as described above were ligated with T4DNA ligase to transform E. coli C600. Plasmid was extracted from the tetracycline-resistant transformant to obtain PTRP G13.
  • the restriction enzyme EcoRI [produced by Takara Shuzo Co., Ltd.] was added to 50 zg of the plasmid pHBr330 prepared by the method of Reference Example 1 described in Japanese Patent Application Publication No. And BamH I in a 100 / ⁇ reaction mixture [100 mM Tris-5 HC1 ( ⁇ 7.5), 7 mM MgC 12 > ⁇ 0 mM ⁇ a C 1, 7 mM 2-mercaptoethanol ]
  • a 100 / ⁇ reaction mixture [100 mM Tris-5 HC1 ( ⁇ 7.5), 7 mM MgC 12 > ⁇ 0 mM ⁇ a C 1, 7 mM 2-mercaptoethanol ]
  • Electrophoresis was performed at 140 V for 2 hours in a buffer [100 mM Tris-HCI, 100 mM boric acid, 2 mM EDTA (pH 8.3)] using a lab gel. After the electrophoresis, a 1.4 kb DNA fragment was separated from the gel
  • the reaction solution was used to transform Escherichia coli 294 strain, and a plasmid PHBrP31—17 in which the BamHI site of the plasmid pHBr31 was converted to a PstI site was separated (FIG. 5). reference).
  • the plasmid pTRP P31 -R, 400 was mixed with 100 units of the restriction enzyme Clal and PstI at 800
  • the 1.42 kl) DNA fragment and 50 g of 50 uL of a restriction enzyme BstN I were added to 100 g of a reaction solution of 100 ⁇ [10 mM Tris-HCl (pH 8.0O). ), 20mM KC 1, 6 mM C 1 2 6 mM 2- Melka 'but-Etano - after act 6 0 ° C, 1 5 min in Le] immediately Fuweno - were deproteinized Le.
  • the reaction solution was subjected to electrophoresis using 1.5% agarose-slab gel under the above conditions. After the electrophoresis, BstNI partially digested 6355 bp and 683 bp DNA fragments were separated from the gel by the method described in Example 3 (see FIG. 6).
  • the reaction was carried out in exactly the same manner as above except that the 63 ⁇ bp DNA fragment in the above reaction solution was replaced with the above 683 bp DNA fragment, and the E. coli 294 strain was transformed with the reaction solution. Plasmid with three types of DNA bound from transformants
  • PTRP P31-22-27 was obtained (see FIG. 6).
  • This plasmid encodes an adr-type HBsAgP31 peptide in which the 227th tributofan is the C-terminal (see Fig. 7).
  • Example 6 Expression of the HBsAg P31 gene and its fragment in Escherichia coli
  • Each transformant containing the expression plasmid of the HBsAg P31 gene and its fragment obtained in Example 5 was subjected to 2.0% glucose, 1.0% After culturing at 37 ° C for 8 hours in M-9 medium containing acetic acid, the cells were collected and buffered.
  • the plate was washed with [30 mM Tris-HCl (pH 8.0), 30 i NaC 1,5 tnM EDTA].
  • the cells were suspended in a lysate consisting of 10 mM Tris-HCl ( ⁇ 8.0), 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 5 mg Zml lysozyme, and lysed. Guanidine hydrochloride was added to the lysate to a final concentration of 7M, and the mixture was incubated at 37 ° C for 2 hours.
  • the lysate was centrifuged at room temperature at 15,000 rpm for 15 minutes to obtain a supernatant. An aliquot of this supernatant was spotted on a piece of filter paper activated with cyanogen bromide. This piece of filter paper was added to a 5% glycine solution [5% glycine, 50 mM Tris-
  • Escherichia coli C600 is designated as IFO-144 by the Fermentation Research Institute (IF0) and FERMP-815 by the Institute of Microbial Technology (FRI) of the National Institute of Advanced Industrial Science and Technology.
  • Escherichia coli C600 / TR ⁇ G7 has been deposited as IF0—'144430 in IF0 and FERMP—8143 as Ffil and deposited in Escherichia coli C6.
  • 0 0 / TR ⁇ Gl 2 has been deposited in the IFO as IFO—144 4 31 and in FRI as FERM P—8 14 2.
  • Escherichia coli C 600 / pT R ⁇ SS — 6 is IFO Deposited as IFO-1442 9 and FRI as FERMP-8144
  • Escherichia coli 294 / pTR P P 31 -R has been deposited with IF 0 as IF 0 -144 5 55 and with F RI as FERMP-7709.
  • Escherichia coli 294 is a known bacterium [Proc. Natl. Acad. Sci. USA 73, 4174 (1974)], and has been deposited with the IFO as IFO-141171. Have been.
  • novel protein produced according to the present invention is useful as a vaccine useful for diagnosis of HBV and prevention of HBV infection.

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Abstract

Un antigène de surface du virus de l'hépatite B présentant une déficience partielle sur le côté terminal C est obtenu par culture d'un transformant contenant un ADN recombinant codant l'antigène de surface du virus de l'hépatite B, lequel ADN est partiellement défectueux sur le côté terminal 3 et possède une région d'activation sur son côté amont et un codon d'arrêt sur son côté aval.
PCT/JP1985/000161 1985-04-03 1985-04-03 Nouvel adn et son utilisation WO1986005808A1 (fr)

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PCT/JP1985/000161 WO1986005808A1 (fr) 1985-04-03 1985-04-03 Nouvel adn et son utilisation
JP409286A JPS61231997A (ja) 1985-04-03 1986-01-10 新規ｄｎａおよびその用途

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2006272427B2 (en) * 2005-07-18 2008-05-22 Gilbert Alain Lindsay Garrick Method of facilitating access to operator functions of hazardous condition alarm devices

Citations (1)

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

Patent Citations (1)

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

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2006272427B2 (en) * 2005-07-18 2008-05-22 Gilbert Alain Lindsay Garrick Method of facilitating access to operator functions of hazardous condition alarm devices
AU2006272427B8 (en) * 2005-07-18 2008-06-26 Gilbert Alain Lindsay Garrick Method of facilitating access to operator functions of hazardous condition alarm devices

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