• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    A recombinant plasmid inserted with Hepatitis B virus gene, which comprises a plasmid vector containing a yeast gene and an E. coli gene 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 culturing 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 human blood plasma and is useful for the preparation of Hepatitis B virus vaccine and diagnostic reagents.
    公开号:SU1387878A3
    申请号:SU833635804
    申请日:1983-08-15
    公开日:1988-04-07
    发明作者:Миянохара Ацуси；Нозаки Сикатеру；Хамада Фукусабуро；Тох-Е Акио；Охтомо Нобуяа；Мацубара Кениси
    申请人:Джуридикал Фаундейшн Дзе Кемо-Серо-Терапевтик Рисерч Институт (Фирма)；
    IPC主号:
    专利说明:

    with
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    This invention relates to the field of biotechnology and relates to the preparation of the hepatitis B virus surface antigen protein.
    The purpose of the invention is to increase the yield of the final product.
    The production of HBsAj is ensured by using recombinant DNA polymerase of the single-stranded region of the DRSh, which is extended to fully double-stranded, and a labeled (32P) material is obtained. This material is added to the upper part of the centrifugation tube, in which the layers contain 30, 20, and 10% solutions of sanatorium plasmid DNA in the specified order, and the tube is centrified. As a result of the cultivation of Saccharorayces cerevisiae a transformed plasmid that contains the marker gene for E, coli and yeast.
    In order to destroy the enzyme proteins firmly bound in, DNA
    and can replicate as in E. coli, 15 the resulting precipitates are treated in
    20
    and in yeast, while the HBsAg antigen gene in this plasmid is inserted downstream from the acid phosphatase promoter, in which part or the entire structure of the acidic phosphatase gene — or some region upward from the structural acid phosphatase gene.
    Example 1. Collect blood plasma (700 ml) taken from ten patients with a positive reaction to 25 HBsAg (adr subtype) and to HBsAgj and then subjected to centrifugation at 5000 rpm for 20 minutes to remove undissolved materials. The resulting solution was centrifuged at 4 ° C at a rate of 18,000 rpm for 8 hours, and the resulting precipitate was redissolved in 10 ml of buffer (pH 7.5) 10% Tris-HCl, 0.1 M NaCl and 1 mM EDTA. The resulting solution is added to the top of a centrifugation tube containing 30% sucrose, which is centrifuged at 39000 rpm for 4 hours. The resulting precipitate is redissolved in the same buffer.
    In order to facilitate subsequent operations, the buffer solution is subjected to 45 reactions with the hepatitis B virus polymerase using its treatment in a mixture (500 μl) of 67 mM Tris-HCl (pH 7.5), VO mM, 25 mM MgClg., 0.5 % NP40 (tergitol, manufactured by Sigma Co.), 0.1% 2-mercaptosanthanol, 330 μMdDTP (deoxycytidine triphosphate), dGTP (deoxyguanosine trisbosLata) and dATP (deoxyadenosine
    35
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    a mixture of 1 mg / ml of pronase E, which was produced by Kaken Kagaku K. and a 0.2% aqueous solution of laurel: sodium sulfate at 3 ° C for 2 hours. The resulting mixture is extracted twice with phenol (200 Oe. The resulting extract is DNA-containing, washed with ether to remove the phenol, and then DNA solution is obtained in the hepatitis B rus. The DNA obtained this time has a specific radio activity of 2.5 x 10 counts / min
    . 1 ISS.
    The obtained double-stranded circular DNA of the hepatitis B virus (HBV) is a clone using the DNA of the A-phage Sharo 16A as a vector, and then cloned again into the plasmid pACUS1
    HBV DNA (20 ng) is treated with Xho 1 nuclease in a mixture (20 μl) of 10 mM Tris-HCl (pH 7.4), 7 mM M 100 mM NaCl, and 7 mM 2-mercaptoethane at 37 ° C for 2 h. Obtained As a result, the mixture is extracted with a hairdryer (20 µl) and then with ether, a double volume of cooled ethanol is added to the aqueous layer so that the DNA precipitates. The mixture was kept at -70 ° C for 1 hour, and then centrifuged at 10,000 rpm for 5 minutes, and the DNA was extracted. The precipitates thus obtained are separated and dissolved in a mixture (5 L) of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. HBV DNA and p nomolar amount of L-phage ron 16A DNA (having one site for Xho I) obtained as a result of
    triphosphate, O, 5 µM (P) dTTP (dissection endonuclease dissection section
    oxythymidine triphosphate) for 3 h and the same volume of 100 i EDTA solution is added to the reaction mixture. As a result of the above reaction with Xho I, the same method as used above was treated with a T4 DNA ligature mixture of 50 mM Tris-HCl (pH 7 10 f MgCl, 10 mM dithiothreitol.
    With the help of DNA polymerase, the single-stranded region of DRSh is extended to completely double-stranded, and material labeled (32P) is obtained. This material is added to the upper part of the centrifugation tube, in which 30, 20 and 10% sucrose solutions are contained in layers in this order, and the tube is centrifuged at 4 ° C at 39,000 rpm for 4.5 hours. .
    In order to break down the enzyme, the proteins firmly bound in, DNA,
    the precipitates obtained are treated in
    0
    50
    five
    five
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    a mixture of 1 mg / ml pronase E, which is manufactured by Kaken Kagaku K.K. and 0.2% aqueous solution of sodium lauryl: sodium sulfate at 37 ° C for 2 hours. The resulting mixture is extracted twice with phenol (200 L), E Poluchenny in. As a result, the extract containing DNA is washed with ether in order to remove phenol, after which the DNA solution of the hepatitis B virus is obtained. The DNA thus obtained has a specific radioactivity of 2.5x10 counts / min per
    . 1 ISS.
    The resulting double-stranded circular DNA of the hepatitis B virus (HBV) is cloned using the DNA of the A phage Sharon 16A as a vector, and then cloned again into the plasmid pACC177.
    HBV DNA (20 ng) is treated with Xho 1 endonuclease in a mixture (20 μl) of 10 mM Tris-HCl (pH 7.4), 7 mM MgClI, 100 mM NaCl, and 7 mM 2-mercaptoethanol at 37 C for 2 h The resulting mixture is extracted with phenol (20 µl) and then ether, and a double volume of chilled ethanol is added to the water layer so that the DNA precipitates. The mixture is kept at -70 ° C for 1 h, and then centrifuged at 10,000 rpm for 5 minutes, and the precipitated DNA is recovered. The precipitates thus obtained are separated and dissolved in a mixture (5 L) of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. HBV DNA and a uniformly equal amount of L-phage Sharon 16A DNA (having one recognition site for Xho I), the resulting
    with dissection endonuclease section
    Xho I, by the same method as used above, was treated with a T4 DNA ligase mixture of 50 mM Tris-HCl (pH 7.4), 10 f MgClI, 10 mM dithiothreitol.
    10 μg / ml albumin of calf serum, 0.5 mM ATP and 0.5 μl ligazon TA produced by Ticar Biomedicals, 1-5-10 units / ml at 4 ° C for 18 hours. The reaction mixture is extracted phenol and simple ether, and then subjected to precipitation with phenol. The precipitates thus obtained are dissolved in a mixture (10 µl) of 10 mM TrisH1 (pH 7.4) and 1 mM EDTA.
    The recombinant DICK is packaged and D-phages are obtained, which then on a flat L-arape (23x23 cm) form plaques (10) using E. coli SupF DR50 as an indicator. Such blasts are hybridized using as probe.
    Labeled HBV DNA
    32,
    So as to
    it was possible to isolate blp: and, formed by a phage containing HBV DNA. Phage DNA is prepared using DRi-SupF E.i-.oli as an infectious bacterium. The DNA thus obtained is treated with Xho I under the same conditions as described for 2 hours and the resulting reaction mixture is subjected to electrophoresis using a 0.75% agar gel to isolate HBV DNA (3.2 Kb). HBV DNA is absorbed onto DEAE (diethylaminoethyl cellulose - paper, manufactured by Toyo Roshi, Japan) in order to separate the carrier DNA, and then eluted with 1 M aqueous NaCl solution in order to obtain HBV DNA having Xho 1 sites on both sides.
    Separately, the plasmid raCUS17-7, containing the only site of the Xho 1 section within its kanamycin resistance gene, is treated with Xho 1, and the product is purified by means of extraction with phenol, treatment with ether, and precipitation with ethanol. Thus obtained plasmid RASUS177, dissected by the enzyme Xho 1 HBV DNA in a molar ratio of 1: 5, and the mixture is stitched DNA T4-ligase for 1H hours
    Isolation of DNA (10 µl) is carried out as follows: the culture broth of strain E is processed. co.1 i v1776, this mixture is thoroughly mixed and maintained at for
    55
    25 min The mixture is placed on an L-agar plate containing ampicillin (20 μg / ml), o (α-biotin (1 μg / ml), diaminopimelic acid (100 μg / ml)
    o
    five
    0 5
    0
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    0
    five
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    five
    and thymine (20 µg / ml) and injected at 37 ° C. overnight. The resulting colonies are placed on an agar plate containing kanamycin (20 µg / ml), on an agar plate containing ampicillin (20 µg / ml), and colonies that grow only on an agar plate containing ampicillin are selected for rUSUS177 containing the resistance gene ampicillin and the kanamycin resistance gene, but when the HBV DNA is inserted into the Xho 1 site of the kanamycin resistance gene, it loses the kanamycin resistance property. Accordingly, the selected colonies contain the HBV recombinant RASUS177-DNA. From the colonies thus selected, a plasmid is obtained, i.e. The recombinant DNA, pACUS177 - HBV DNA (which is referred to as pHVU), is treated with the enzyme Xho 1, resulting in the entire HBV DNA fragment (3.2 kB). When the plasmid is treated with the enzyme Xho 1 and the enzyme BamH1, a fragment (approximately 1.3 kb) containing the HBsAg gene is obtained.
    An EcoRI fragment of approximately 8,000 nucleotide pao (8 kb) containing a 60,000 dalton polypeptide gene (PbO), which constitutes the acid phosphatase gene under repression, is obtained from the yeast S288C gene bank and inserted into the EcoRI site of the plasmid pBR322 of E. coli whereby the original plasmid is obtained. .
    The original plasmid is treated with the Sail endonuclease and stitched with DNA - T4 ligase, thus obtaining the plasmid pAT25, which has a gap from the Sal 1 site to a fragment of the 5.2 kb acid phosphatase gene. The pAT 25 plasmid is a plasmid containing its fragment (approximately 3.7 kb) from the EcoRI site to the Sal I site of pBR322, which contains the ampicillin resistance gene and the fragment (approximately 2.8 kb) from the EcoRI site to the Sal 1 gene yeast acid phosphatase.
    In EcoRI, the pAT 25 site is inserted with an EcoRI fragment (1.4 kb) containing ars 1 and the Trp 1 gene, which is obtained by treating the plasmid yRP 7 with an EcoRI enzyme, thus obtaining the plasmid pAT 26. The fragment ars 1-Tr p 1 contains the only site indicated for the enzyme is the Hind III cross section — inside the Trp1 gene.
    The pAT 26 plasmid is inserted into the Hind III site of a Hind III fragment containing Len 2 and 2 jU ori, which is obtained by treating the plasmid pSLE 1 with the Hind III enzyme in order to obtain the shuttle vector pAT, 77. rAT 77 contained in S.cerevisiae (i.e. Saccharorayces cerevisiae AH 22 (pAT 77)) was deposited with the Research Institute of Fermentation, Japan Science and Technology Agency, according to the Budapest Agreement FEPM BP-324) ..
    Thus, rats 77 (1 μg) are dissected with the enzyme Sal 1, and then treated with exonuclease BAL 31 (0.1 and) in a solution (50 μl) with 20 mM Tris-HCl (pH 8.2), 12 mM CaCl, 12 mM MgCl2, 0.2 M, NaCl and 1 mM EDTA for 30 s to 1 min. The reaction mixture is subjected to extraction with phenol and precipitation with ethanol. The resulting precipitates are treated with Xho 1 linker (1 nmol) and DNA with T4 ligase under the same conditions as described for 12 hours.
    The transformed E. coli X.1776 plasmid was obtained and a transformed ampicillin resistant culture was obtained. Plasma MIDA DNA was extracted from the colonies, the nucleotide sequence of the DNA was determined, and then the region of the acid phosphatase gene was deleted using BAL 31. DNAs are selected and the pAM 81, pAM 82, pAM 83 and pAM 84 plasmids are found that lack the structure of the phosphatase gene.
    Denote A in the codon ATG ,. encoding the first amino acid.slot (methionine) of the PbO product of the structural phosphatase gene, the following areas are removed via shuttle carriers; pAM 81: up to +2, pAM 82: down to - 33., pAM 83: down to -50 and pAM 84: up to - 51. pAM 8l, pAM 82, pAM 83 and pAM 84, contained in Saceharorayces serevisiae- (i.e. Carpentry cereumia cereusia AH 22 (pAM 81, AH 22) pAM 82, AH 22 (pAM 83 and AH 22) pAM 84, - deposited in the Research Institute of Fermentation,. The Agency for Industrial Science and Techno

    login. Japan, in accordance with the Buda Pest Agreement FEPM BP-325, FEPN BP-313, FEPM BP-327 and FEPM BP-326.
    ten
    15
    20
    25
     .,. thirty
    35
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    As a result of treating the pHBU plasmid (pACCUS 177-HBV DNA) with Xho 1, the creeped HBV DNA is subjected to recombination with the Shuttle Vector, dissected with Xho 1, pAM 81, pAM 82, pAM 83 and pAM 84 in a molar ratio of 5: 1 using DNA crosslinking by T4 ligase. .
    E. coli X 1776 is transformed with this plasmid DNA, ampicillin-resistant colonies are obtained from the transformed culture. DNA is isolated from them and analyzed using cross-section enzymes such as Xho 1, Xha 1 and Hind III, thus identifying the insertion of the BHV DNA into carriers and its direction.
    . A fragment of the HBsAg gene (3 µg), resulting from the dissection of the pHBU plasmid with the HBH1 enzyme, is treated with DNA polymerase T4 (0.2U) in a solution (100 µl) of 67 i Tris-HCl (pH 8.6), 6 , 7 mM MgCl2, 10 mM 2-mercaptoethanol, 6.7 μM EDTA and 16.7 mM (W4) s804, which contain 200 μM o.ATP, as.CTP, XTTP and ILGTP for 30 minutes so that fill the end of the cross section with the enzyme BacH1. - The reaction mixture is extracted with phenol and precipitated with ethanol. The precipitates are crosslinked with Xho linker, 1 at a molar ratio of 1:10 using T4 DNA ligase. After extraction with phenol and precipitation of the plasmid with ethanol, the plasmid is treated with Xho 1, thereby obtaining a fragment of the HBsAg-gene (approximately 1.3 kb) containing the terminal of the Xho 1 section at both ends. This fragment is stitched with the shuttle vector pAM 82, which is dissected with the Xho .1 enzyme, in a molar ratio of 5: 1 using T4 ligase DNA, transforming E. coli X 1776 with E. coli recombinant DNA obtained.
    The HBsAg gene is inserted into this plasmid in the correct direction downward from the phosphatase promoter of the pAM 82 vector,
    The starting yeast is Sacefaromyces cerevisiae AN22 (a, len 2, his 4, caul (Cir)), which have been deposited at the Research Institute of Fermentation, Industrial Science and Technology Agency, Japan, in accordance with the Budapest Agreement under the name FEPM BP-312. The original yeast is placed on an URV medium (-100 ml) containing 2% polypeptone, 1% yeast extract, and 2% glucose, then a mixture
    incubated at 30 ° C overnight and then the cells are harvested by centrifugation. The cells thus collected are washed with sterilized water (20 ml), suspended in a solution of 1.2 M sorbitol and 100 µg / ml zymolyase -60000 (which is manufactured by Seikagaku Kogio K, K., Japan), the suspension is kept for 30 min, resulting in a spheroplast. The spheroplast thus obtained is flushed with a 1.2 M solution of sorbitol three times, and then suspended in a solution (0.6 ml) of 2 M sorbitol, 10 mM CaCl and 10 mM Tris-HCl (pH 7.5). Suspension realizpot in small tubes in a volume of 60 μl. A solution of the recombinant plasmid PAN 203 (30 µl) is added to the suspension. After thorough mixing, the mixture is added with 0.1 M CaCl (3 µl) to room concentration of CaC in 10 mM, then the mixture is held at room temperature for 5-10 minutes. B, the resulting mixture of 1 ml of a solution of 20% polyethylene glycol 4000, 10 mM CaCl and 10 mM Tris-HCl (pH 7.5), then the mixture is kept at room temperature for about 20 minutes. The mixture (each with a volume of 0.2 ml) was added to a medium (10 ml) consisting of 22% sorbitol, 2% glucose, 0.7% is amino acid based on yeast nitrogen, 2% yPD , 20 mg / mp from histidine and 3% from agar and kept at a constant temperature of 45 C. After vigorous stirring, the mixture is placed in a layer on a plate with minimal medium containing 1.2 M sorbitol, which is prepared in advance it contains 0.7% amino acid based on yeast nitrogen, 2% glucose, 20 μg / ml histidine and 2% agar. Incubirztot plate, resulting in a yeast colony it does not depend on the boiling leudina.Koloniyu incubated in minimal medium Burke Hol- dera supplemented with histidine (20 mg / ml; to give the desired transformed yeast: Saceharomy .ces cerevisiae 203 the RAS.
    : In the same way, however, instead of recombinant RAN 203, recombinant plasmids are used, PAS 101, RAN 201 and RAN 205; receive the following transformed yeast: Saceharomy
    ces cerevisiae pAS 101, S. Cerevisiae pAH 201, S. Cerevisiae pAH 205,
    Each colony of transformed carrots was applied to an agar plate with a minimum Berk Holder medium filled with histidine (20 µg / ml) and incubated before the formation of a colony. The resulting cells are separated from the colony, inoculated onto a minimal Berk Holder medium, additional histidine (20 µg / ml) and incubated at 30 ° C. After about 24 hours, cells in the logarithmic growth stage are harvested by centrifugation, suspended in minimal) medium (.10 ml), but containing phosphoric acid (which is prepared by replacing KHjP04 in Berck Holder minimal medium with KC1 and then adding 20 µg / ml histidine), with con /
    a cell concentration of approximately 4x10 cells / ml. After incubation at 30 ° C for about 24 hours, the culture broth is centrifuged at 4000 rpm for 10 minutes to collect the cells. The cells thus collected are suspended in a. a solution (3 ml) of 1.2m sorbitol, 50 mM phosphate buffer (pH 7.2), 14 1 2-mercaptoethanol, and 100 g / ml Zymolyase - 60,000 (manufactured by Seyakagaku Kogyo K.K., Japan), and then the mixture vigorously Shake at 30 minutes to obtain a spheroplast. Spheroplast is collected by centrifuging and carefully suspended in a solution (1 ml) of 0.1% Triton X-100 and 50 mM phosphate buffer (pH 7.2), the suspension is vigorously stirred, and then centrifuged at 7,000 rpm for 10 min, while the top layer forms a solution of lysed yeast.
    The resulting solution (20 μl) is. Using an RIA anti-HBS antigen device (manufactured by Abbott, U.S.A.) for HBS antigen activity. The results are shown in the table.
    As for the solution of lysed yeast obtained from S.cerevisiae AH 22 / PAN 203, it is assumed that the activity and amount of antigen are in accordance with a static parallel subfield when using a device for the detection of HBaAg, and purified HBsAg obtained from human serum.
    A dosed solution (each portion of 0.4 ml) was administered to Guinean pigs (females, 300-380 g, 10 animals) once a week for three weeks by the subcutaneous method, and the antibodies in plasma ™ were determined using for detecting anti-HBS antibodies (AUSAB, manufactured by Abbott, U.S.A.). As a result, all animals found ITI-NVZ antigenera. Formula of the invention 1. A method for producing a hepatitis B virus surface antigen protein, including designing a shuttle-20 vector by combining a Saceharomyces cerevisiae DNA fragment containing ars 1,2pori and a selective marker for selecting transformed . S.cerevisiat; with the Esche-25 richia coli DNA fragment containing the plasmid replication gene, as well as a selective marker gene for selection of transformed E. coli plasmid, introducing into it a portion of the DNA encoding the hepatitis B surface antigen (HBsAg), which is under the control of a yeast promoter , followed by cultivation of the transformed plasmid SoCerevisiae, isolation and purification of the target product, characterized in that, in order to increase the yield of the final product, the construction of the vector is carried out by introducing into the E.coRI site
    4U
    Plasmid pBR322 of the acid phosphatase gene P60, derived from the gene bank of the S288C yeast, is further treated with the Sal I nucleonuclease and DNA ligase
    S. cerevisiae
    AH 22 (FEPM) RAN 201 10.597 2- RAN 203 13, .008
    -
    T4 and get the plasmid pAT25, include in Sal I the plasmid pAT25, the arsl-trpi fragment, from plaskid 4P7, the obtained plasmid pAT 26 is treated with the Hind III nucleonuclease, and the fragment Len 2 and 2 // the plasmid pS2F is introduced, after which the resulting vector pAT77 and a fragment containing the HB3Ap gene; The subtype isolated from the pHBV plasmid with the aid of the Wat HI fragment is treated with Xho I endonuclease, the obtained fragments are DNA-ligated T4 and the recombinant plasmid DNAs are selected in which the HBsAg gene is located below the promoter region
    2, the method according to claim 1, distinguishing from that the vector pAT77 is cleaved with the endonucleases Sail and Bgl 31, the Xho I linker is added and the resulting mixture is treated with T4 DNA ligase, the nucleotide sequence is determined from the resulting plasmids and choose the plasmid pAI up to +2, pAM from 82 to -33, pAM 83 to -50 and pAM 83 to -51, from which part of the PbO gene is removed.
    3, the method of claim 1, wherein the blood plasma of patients with hepatitis B isolates the virus virus, the single-strand DNA is doubled with DNA polymerase 1, the resulting DNA is treated with Xho 1 endonuclease and cloned into the Xho 1 DNA site of pharon 15A , isolated by hybridization with labeled DNA of hepatitis B virus, phage DNA and PLAS / 77 PL-azmide are treated with Xho 1 with endonuclease, the resulting fragments are stitched with DNA ligase T4, then transformed with the obtained recombinant DNA of E. coli cell and the recombinant pH-plasmid is transformed containing the gene HBsAg
    ran 205 5.548
    ch138787812
    Table continuation
     This carrier contains neither the Bt B gene nor the HBS gene and is used as a negative control clone (a negative control clone using an RIA device has an activity of 310 counts / min, and a positive control clone has the same indicator of 17,500 counts / min.
    RA 101 P.200
    权利要求:
    Claims (3)
    [1]
    Claim
    1. A method for producing hepatitis B virus surface antigen protein, * comprising constructing a shuttle-20th vector by combining a Saceharomyces cerevisiae DNA fragment containing ars 1,2pori and a selection marker for selection of transformed ones.
    S. cerevisiae with the Esche-25 richia coli DNA fragment containing the plasmid replication gene, as well as a selective marker gene for selection of E. coli transformed with the plasmid, introducing into it a portion of the DNA encoding the hepatitis B surface antigen gene (HBsAg), which is controlled yeast promoter, followed by culturing the transformed with the plasmid S o cerevisiae, isolation and purification of the target product, characterized in that, in order to increase the yield of the final product, the construction of the vector is carried out by introducing into the E.coRI site
    4 and plasmids pBR.322 of the gene for acid phosphatase P60 isolated from the gene bank of S 288C yeast, then treated with Sal I endonuclease and DNA ligase
    T4 and receive the plasmid pAT25, include in the Sal I plasmid pAT25 the arsl-trpl fragment isolated from the t 4P7 plasmid, the resulting plasmid pAT 26 is treated with the Hind III endonuclease, and the Len 2 and 2 uori fragment of the plasmid pS2F is introduced, after which the resulting pAT77 vector and the fragment containing the HBsAg gene of subtype ad * g, isolated from the pHBV plasmid using the Bam HI fragment, treated with Xho I endonuclease, the obtained fragments are crosslinked with T4 DNA ligase, and recombinant plasmid DNAs in which the HBsAg gene is located below the promoter region are selected
    [2]
    2. The method of pop. 1, characterized in that the pAT77 vector is dissected with Sall and Bgl 31 endonucleases, an Xho I linker is added and the resulting mixture is treated with T4 DNA ligase, the nucleotide sequence is determined from the obtained plasmids, and the plasmids pAM to +2, pAM from 82 to -33, pAM 83 to -50 and pAM 83 to -51, from which a portion of the P60 gene has been deleted,
    [3]
    3, the way pop. 1, characterized in that · the blood plasma of patients with hepatitis B secrete virus DNA, single-stranded DNA is doubled with DNA polymerase 1, the obtained DNA is treated with Xho 1 endonuclease and the Sharon 15A Λ phage DNA site is cloned into Xho 1, isolated by hybridization with labeled with hepatitis B virus RNA, phage DNA and pCUS / P plasmid are treated with Xho 1 endonuclease, the obtained fragments are cross-linked with T4 DNA ligase, then E. coli cells are transformed with recombinant DNA and the recombinant pHP4 plasmid containing the HBsAgo gene is selected
    Clone Master Plasmid HBsAg activity. 1 IΊ B 4
    S. cerevisiae
    AN 22 (FEPM BP-312) RAN 201 10.597
    2 - RAN 203 13.008 RAN 205 5.548
    1387878 12
    Table continuation
    1 I
    1 2 S 4 4 - - pA 101 11.200 ControlRAM 82 * 320
    * This carrier contains neither the bb gene nor the HBS gene and is used as a negative control clone (a negative control clone using an RIA device has an activity of 310 counts / min, and a positive control clone has the same rate of 17,500 counts / min.
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    同族专利:
    公开号 | 公开日
    ES540895A0|1988-04-01|
    DK169953B1|1995-04-10|
    ES8600059A1|1985-10-01|
    JPS6155950B2|1986-11-29|
    US4778761A|1988-10-18|
    KR840005745A|1984-11-15|
    ES8802162A1|1988-04-01|
    KR890000096B1|1989-03-07|
    ES524942A0|1985-10-01|
    CA1270217A|1990-06-12|
    DK372183A|1984-02-17|
    IL69423D0|1983-11-30|
    DK372183D0|1983-08-15|
    JPS5931799A|1984-02-20|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    LT3988B|1992-02-17|1996-06-25|Fermentas Biotech Inst|Recombinant plasmides pfs19, pfps2-48 and pjlfds1 codingsynthesis of human hepatite b of surfice virus antigenes, methods fof producing thereof|FR2444713B1|1978-12-18|1981-02-06|Pasteur Institut|
    FR2480779B2|1979-08-30|1986-07-18|Anvar|VECTOR CONTAINING A NUCLEOTIDE SEQUENCE OF THE SURFACE ANTIGEN OF HEPATITIS B VIRUS AND METHOD FOR MANUFACTURING AN IMMUNOGENIC MOLECULE USING THE SAME|
    US4615974A|1981-08-25|1986-10-07|Celltech Limited|Yeast expression vectors|
    US4446235A|1982-03-22|1984-05-01|Genentech, Inc.|Method for cloning human growth hormone varient genes|
    US4546082A|1982-06-17|1985-10-08|Regents Of The Univ. Of California|E. coli/Saccharomyces cerevisiae plasmid cloning vector containing the alpha-factor gene for secretion and processing of hybrid proteins|
    GB2125047B|1982-08-09|1986-02-19|Ciba Geigy Ag|Yeast hybrid vectors and their use for the production of polypeptides|
    AU584580B2|1982-09-08|1989-06-01|Smith Kline - Rit|Hepatitis B virus vaccine|
    CA1341116C|1983-02-22|2000-10-17|Rae Lyn Burke|Yeast expression systems with vectors having gapdh or pyk promoters and synthesis or foreign protein|GB2125047B|1982-08-09|1986-02-19|Ciba Geigy Ag|Yeast hybrid vectors and their use for the production of polypeptides|
    JPS6155951B2|1982-08-20|1986-11-29|Kagaku Oyobi Ketsuseiryoho Kenkyusho|
    JPH0543352B2|1982-10-19|1993-07-01|Takeda Chemical Industries Ltd|
    JPH0552190B2|1984-12-11|1993-08-04|Chemo Sero Therapeut Res Inst|
    US4816564A|1986-01-31|1989-03-28|Merck & Co., Inc.|Method for producing hepatitis B virus proteins in yeast|
    US5190877A|1987-09-03|1993-03-02|Gist-Brocades N.V.|Saccharomyces strains for maltose fermentation|
    US4883865A|1987-09-30|1989-11-28|Merck & Co. Inc.|Recovery of pres2+s antigen|
    AP129A|1988-06-03|1991-04-17|Smithkline Biologicals S A|Expression of retrovirus gag protein eukaryotic cells|
    US6319501B1|1990-07-19|2001-11-20|Health Research, Inc.|Method of immunizing against hepatitis B virus|
    US5531990A|1993-12-15|1996-07-02|Health Research, Inc.|Method of raising an immune response with an anti-idiotypic antibody having correspondence with human hepatitis B surface antigen|
    IL118626D0|1996-06-11|1996-10-16|Xtl Biopharmaceuticals Limited|Anti HBV antibody|
    US6451572B1|1998-06-25|2002-09-17|Cornell Research Foundation, Inc.|Overexpression of phytase genes in yeast systems|
    AU4056700A|1999-03-31|2000-10-16|Cornell Research Foundation Inc.|Phosphatases with improved phytase activity|
    US6841370B1|1999-11-18|2005-01-11|Cornell Research Foundation, Inc.|Site-directed mutagenesis of Escherichia coli phytase|
    WO2003037102A2|2001-10-31|2003-05-08|Phytex, Llc|Phytase-containing animal food and method|
    AU2003272410A1|2002-09-13|2004-04-30|Cornell Research Foundation, Inc.|Using mutations to improve aspergillus phytases|
    US7919297B2|2006-02-21|2011-04-05|Cornell Research Foundation, Inc.|Mutants of Aspergillus niger PhyA phytase and Aspergillus fumigatus phytase|
    US8540984B2|2006-08-03|2013-09-24|Cornell Research Foundation, Inc.|Phytases with improved thermal stability|
    WO2008136790A1|2007-05-02|2008-11-13|Merial Limited|Dna plasmids having improved expression and stability|
    US8192734B2|2007-07-09|2012-06-05|Cornell University|Compositions and methods for bone strengthening|
    CN112680464A|2020-12-10|2021-04-20|南京农业大学|Monomethylarsenic and trivalent antimony oxidase gene arsV, and protein coded by same and application thereof|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    JP57142460A|JPS6155950B2|1982-08-16|1982-08-16|
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