LT3988B - Recombinant plasmides pfs19, pfps2-48 and pjlfds1 codingsynthesis of human hepatite b of surfice virus antigenes, methods fof producing thereof - Google Patents

Abstract

The yeast Saccharomyces cerevisiae, which produces huge quantities of the surface antigens of the human virus of the B hepatitis HBsAG, HBpreS2Ag and HbpreS1Ag are constructed by using methods of gene engineering. The effective biosynthesis of antigens in the yeast was achieved by the use of the hybrid promoter of the yeast, which consists of the promoter parts of the genes GAL1 and PYK1. For the stabilization of the recombinant plastids and the selection of the productive stems, a gene from the yeast Candida maltosa was used; it determines the resistance to the formaldehyde. The use of this gene allows growing recombinant stems in the feeding mediums of any of the compositions.

Description

The invention relates to the field of biotechnology and defines a method for obtaining human hepatitis B virus surface antigen producers in Saccharomyces cerevisiae. Hepatitis B virus antigens can be used to develop effective recombinant vaccines and diagnostics.

Most of the known methods for obtaining human hepatitis B virus surface antigens rely on recombinant yeast strains. Yeast is used because it produces 22nm particles of immunologically active protein. These particles are analogous to the 22 nm particles found in plasma of human hepatitis B virus (HBV) carriers.

The synthesis of human hepatitis B virus surface antigens in yeast is achieved by the use of recombinant plasmids with efficient regulatory elements (regulated promoters, transcriptional terminators) and also depends on the recipient yeast strain.

The patents describe the synthesis of a number of plasmids that determine the human hepatitis B virus surface antigens HBsAg, HBpreS1Ag and HBpreS2Ag:

Pyki-6, GAP1-6 et al., According to EP 0164556, int.c. C12N 15 / 00,1985; pHBSGAP 347 / 33T and pHBpreSGAP 347 / 33T according to EP 0174444,1986; pYGAP / pSSA, pYGAP / pSSC, pYGAL / pSSA, pYGAL / pSSC, pYADH2 / pSSA and pYADH2 / pSSC according to EP 0244924, 1987;

pGG6, pGG61 / Tn-1 and pGG63 according to EP 0248410, 1987;

pHBpreSGAP 347 / 19T and pYGpreS2S1 according to EP 0312159.1989;

pT305A and pHBS according to EP 0339567.1989 and EP 0341746.1989;

pPHO P31-R, pPHO P31pGLD P31-R and ρΡΚΤ P31-R by

EP 0401941, 1990.

pYeHBs according to GB 2104902, 1983; pRIT10673 and pRIT10674 according to AU-B-16750 / 83,1983; pAH201, pAH203 and pAH205 by SU 1387878, 1988, and pNMVG 39-54 by SU 1380207.

The use of these plasmids does not provide a highly efficient level of HBV surface antigen synthesis. In addition, the patents do not mention the synthesis of all three proteins, HBSAg, HBpreSIAg, and HBpreS2Ag, found in human plasma HBV carriers in 22nm particles.

The closest method of obtaining human hepatitis B virus surface antigens described herein is EP 0164556, inter. Cl.

C 12 N 15/00, 1985 describes the construction of the Pyk5 plasmid. Plasmid Pyk5 consists of the yeast marker gene LEU2, the 2mkm ori region of the Saccharomyces cerevisiae plasmid, the gene encoding ampicillin resistance and the S gene encoding human hepatitis B virus surface antigen regulated by the hybrid GAL 1-10-PYK promoter and ADH1 transcript.

The following is the construction of Pyk5. Plasmid pLGSD5 was digested with XhoI digestion with Klenow enzyme, ligated with EcoRI linkers (GGAATTCC), then completely digested with EcoRI and Sau3A and isolated 370 b.p. fragment. This fragment was cloned in EcoRI and BamHI digested with pBR322 and the resulting plasmid pBRGAL4.pBRGAL4 was digested with Sau3A restriction enzyme, ligated with SalI linker (CGTCGACG), then treated with SalI and Xhol restriction enzyme. fragment cloned into the Plasmid plasmid restriction site SalI cleavage site to give plot 5GAL4 / 370. The resulting plasmid was digested with BamHI and SalI, the isolated fragment was cloned into pamI / 1GAL4 / 370 treated with pam / 1 and treated with BamHI and SalI. Plasmid plot5 PyksAg57 was digested with SalI and XbaI restriction enzymes, isolated a fragment containing the PYK1 promoter and the 5 'end of the gene and ligated with the XbaI-SalI fragment from the plot 5s AgtADH plasmid containing the S gene 3' end and the ADH1 transcription terminator. The resulting product was digested with restriction enzyme SalI and cloned into the SalI site of plasmid pC1 / 1GAL4 / 370 to obtain plasmid Pyk5.

Principal expression of human hepatitis B virus surface antigens in Saccharomyces carlsbergensis yeast

The disadvantage of the system LTLT 3988 B is the need to use strains containing the leu2 mutation and consequently to use minimal media for culturing these strains containing the recombinant PYK5 plasmid. Yeast strains producing human hepatitis B virus surface antigens grow slowly in such media, with low yields of cellular biomass and recombinant protein. The maximum yield of the immunologically active protein HBsAg is determined by the Pyk5 plasmid to be 1.4 mg / g soluble protein. Maximum productivity in this system using other promoters is 2.1mg / g.

It is an object of the present invention to increase the biosynthesis of human hepatitis B virus surface antigens HBsAg, HBpreSIAg and HBpreS2Ag in S. cerevisiae yeast.

Enhancement of viral antigenic protein biosynthesis is achieved by the use of novel recombinant plasmids pFS19, pFpS2-48 and pJLFdSI encoding HBV surface antigen synthesis, and novel S.cerevisiae strains (FH4C [pFS19], FH4C [pFpS2-48] and FH4C) [beta] 40-80 mg culture of antigenic protein 11. (0.4-0.8% immunologically active protein against total soluble cellular protein).

The recombinant plasmid pFS19, encoding HBsAg protein synthesis, has the following properties:

- is 10.4 thousand b.p. size,

- consisting of:

- a fragment of the plasmid pTZ19R of the Xpnl-Xbal plasmid containing a gene conferring ampicillin resistance and the E. coli ori region;

- a DNA fragment of XbaI-BamHl Candida maltosa containing the FOR-R gene conferring formaldehyde resistance;

- Eco1471 - HindiI fragment containing GAL1-10 transcriptional activator, PYK1 gene promoter, human hepatitis virus S gene, PGK1 gene transcription terminator;

- HindIII-Eco911 2mkm yeast plasmid fragment containing the yeast ori region;

- the FOR-R gene encoding formaldehyde resistance required for selection of transformants;

- have unique BamHI, BglII and SalI recognition sites.

-4EN 3988 B

The recombinant plasmid pFpS2-48, encoding the human hepatitis B virus HBpreS2Ag surface antigen, has the following properties:

- is 10.8 thousand b.p. size;

- consisting of:

A Kpnl-Xbal plasmid fragment of pTZ19R containing a gene conferring ampicillin resistance;

- an XbaI-BamHI Candida maltosa DNA fragment containing the FOR-R gene that confers formaldehyde resistance;

An Ncol-HindIII fragment containing a GAL1-10 gene transcriptional activator, a PYK1 gene promoter, a human hepatitis B virus preS2 gene, and a PGK1 gene transcription terminator;

- HindIII-Eco91I yeast 2mkm plasmid DNA fragment containing the yeast ori region;

- contains the FOR-R formaldehyde resistance gene required for the selection of transformants;

- have unique BamHI, BglII and SalI recognition sites.

The recombinant plasmid pJLFdSI, encoding human hepatitis B virus HBpreSIAg surface antigen synthesis, has the following properties:

- is 11.4 thousand b.p. size,

- consists of:

- Xbal-Pstl C. maltosa DNA fragment containing the FOR-R gene conferring formaldehyde resistance;

An Eco91Ι-HindIII yeast 2mkm plasmid fragment containing the yeast ori region;

- a HindIII-EcoRI fragment containing a transcription terminator of the PGK1 gene, a promoter of the PYK1 gene and a preSl gene of human hepatitis B virus;

An EcoRI-Bsp68I fragment containing the GAL1-10 transcriptional activator gene, the yeast URA3 gene, the ampicillin resistance gene, and the E. coli ori region;

- has a FOR-R gene conferring resistance to formaldehyde;

- have unique BamHI and BglII recognition sites.

The basic principle of the construction of plasmids pFS19, pFpS2-48 and pJLFdSI is the fusion of a FOR-R gene of interest for transformants into a single plasmid

-5LT 3988 B with pre-S1, preS2 or S genes regulated by expression of the hybrid galactose inducible GAL1-10 - PYK1 promoter and PGK1 transcription terminator, and with the 2mkm yeast plasmid ori region and the E. coli gene encoding ampicillin resistance. Cloning of the dominant FOR-R gene for transformant selection involves obtaining a Candida maltosa gene bank using a bifunctional transformation of the yeast vector, yeast Saccharomyces cerevisiae using this LEU2 marker, selection of transformants capable of growing in medium with formaldehyde, plasmid isolation from transformants, subcloning the FOR-R gene and sequencing the primary.

The major steps in constructing human hepatitis B virus surface antigen producers are as follows:

1. Cloning of the dominant transformer FORR gene for formaldehyde resistance.

2. Cloning of the pyruvate kinase-encoding PYK1 and phosphoglycerate kinase-encoding PGK1 genes.

Construction of an expression cassette consisting of the PYK1 and GAL1-10 gene promoter and PGK1 gene terminator.

4. Construction of plasmids encoding human hepatitis B virus surface antigen synthesis in yeast.

5. Search for Saccharomyces cerevisiae strains with reduced proteolytic activity and superproducing superficial human hepatitis B virus surface antigens.

The generation of novel S. cerevisiae strains, superproducers of human hepatitis B virus surface antigens, is based on the transformation of wild-type strains with plasmids pFS19, pFpS2-48 and pJLFdSI using the FOR-R marker gene followed by selection of the best producers among them.

The strain of Saccharomyces cerevisiae FH4C [pFS19] (βκπμ Y1831) producing the surface antigen of human hepatitis B virus has the following characteristics:

Morphological features. Cells oval in shape.

Attributes of culture. Cells grow well on YEPD medium (2% peptone, 2% glucose, 1% yeast extract, 2% agar). Growing on solid media - smooth white colonies that last longer

-6EN 3988 B becomes wrinkled when stored. Grows well in YEPD liquid medium containing 2% peptone, 2% glucose and 1% yeast extract.

Biological-Physiological Characteristics: Cells grow from 10 ° C to 37 ° C, pH optimum 5.0 to 6.0.

It uses glucose, galactose as its carbon source.

Nitrogen source - ammonium salts, peptone, tryptone.

Does not absorb lactose, cellobiose, citric acid, o-xylose, L-arabinose, D-ribose, L-rhamnose.

Resistant to formaldehyde up to 10mM. It has a reduced proteolytic activity.

S.cerevisiae FH4C [pFpS2-48] (βκπμ Y1833) strain producing human hepatitis B virus HBpreS2Ag protein and FH4C [pJLFdSl] (ΒΚΠΜ Y1832) producing HBpreSI protein have similar cultural-morphological and physiological properties.

Figure 1 is a schematic diagram of a cloning gene for formaldehyde resistance. Figure 2 - Nucleotide sequence of FOR-R gene. Figure 3 provides a plasmid containing pPT

Schematic diagram of the PYK1 promoter and construct. FIG. 5,6,7 coding human

Schemes for construction of the transcription terminators of the PGK1 gene, construction of pFpS2-48, pFS19, and pJLFdS1 plasmids of p4pS2-2, Figure 4, for the synthesis of hepatitis B virus surface antigens.

The invention is illustrated by the following examples:

example. Cloning of the dominant FOR-R gene required for transformant selection.

Candida maltosa yeast was grown to late logarithmic phase in YEPD medium and chromosomal DNA was isolated from the cells, chromosomal DNA was partially restricted with Sau3A restriction enzyme, and the resulting fragments were ligated with pL3 plasmid digested Barni restriction enzyme and exposed to alkaline phosphatase. The ligated DNA was used to transform competent Escherichia coli HB101 stem cells. The recombinant DNA was isolated from E. coli and transformed with the C.maltosa gene bank thus obtained into S.cerevisiae DBY746 (his3 leu2 trp1 ura3). After transformation, cells were seeded on minimal medium containing 50mg / ml histidine, uracil and tryptophan. Leu ⁺ transformants were transferred to a complete YEPD medium,

-7LT 3988 B containing 3-10 mM formaldehyde. Transformants of S.cerevisiae growing on formaldehyde medium were selected. When analyzing plasmids isolated from yeast transformants resistant to formaldehyde and re-transformed into E. coli, it was observed that all plasmids contain a common DNA fragment of Candida maltosa. 3.6 thousand b.p. was isolated by subcloning. XbaI-BamHI C.maltosa DNA fragment encoding the FOR-R formaldehyde resistance gene (pRA1 plasmid, Fig. 1). The BamH) -XbaI fragment was sequenced and 1 long open reading frame containing an intron encoding a protein consisting of 381 amino acids was detected (Fig.2). The FOR-R gene, cloned in gauge-containing plasmids containing the 2mkm yeast DNA ori region, confers transformant resistance to 3-10 mM formaldehyde and allows transformation of wild-type S.cerevisiae strains.

example. PGK1 For cloning (phosphoglycerate kinase) and PYK1 gene cloning.

The PYK1 (pyruvate kinase) and PGK1 genes were transformed with S. cerevisiae strain DFY331 (leu2 pgk1) and Pyk1-5 (ade1 leu1 met14 ura3 pyki-5) in S.cerevisiae strain and seeded on synthetic leucine-free media. with glucose as a carbon source. Strains with mutations in anger and pgk1 cannot grow in glucose medium, plasmid DNA was isolated

PYKI plasmids were isolated from the grown transformants and by subcloning).

example, promoter and and PGM genes (Fig.3 pL3-pyk and pL3-pgk from PYK1

Construction of an expression cassette consisting of a PGK1 terminator.

The cloned PGK1 and PYK1 genes have been used as regulatory element sources to construct expression vectors. The pL3-PGK plasmid was restricted with BglII and HindIII restriction enzymes. Excluded 0.38 thousand b.p. The fragment containing the transcription terminator of the PGK1 gene was cloned into the plasmid pIC19R (March et al., Gene, 1984, t32, 481-485p) digested with BglII and HindIII. We called the newly obtained plasmid pPGKT. Plasmid pL3-PYK was restricted to EcoR1 and BglII, yielding 1.0kb. fragment containing the PYK1 gene promoter and ligated with pPGKT plasmid digested with EcoR1 and BglII. We received the pPT

-8LT 3988 B plasmid (Fig.4). The pPT plasmid contains an expression cassette consisting of the PYK1 gene promoter (EcoRI-XbaI) and the PGK1 transcription terminator (BglII-HindIII).

example. Construction of pFpS2-48 plasmid.

mkg plasmid pH320 (ΠγΜπεΗ n _fl p. HAH CCCP, 1983. T.271 c.230-235) was restricted with Dral restriction enzyme (100 units) in 200mL of buffer B (10mM tris-HCl, 10mM MgCl, 1mM DTT; pH 7.50) for 2h. . 37 ° C followed by inactivation of the restriction enzyme by incubation at 70 ° C for 20 min. The DNA was precipitated with 2 volumes of ethanol and centrifuged for 3 min. At 15,000 rpm, DNA was dissolved in 200 mL of ligation buffer (50mM tris-HCl, 10mM MgCl, 10mM DTT; pH 7.5), and adenosine triphosphate (ATP) 0.01 opt. pcs. BglII linkers and 50 pc. T4 DNA ligase, ligated for 16 h. At 12 °.

The resulting DNA mixture was precipitated with ethanol and dissolved in 200 mL of buffer 0 (50 mM Tris-HCl, 10 mM MgCl, 100 mM NaCl, 1 mM DTT; pH

7.5) and affected 50 pieces. BamHI restriction enzymes are 100 units. BglII restriction enzymes 2 or. At 37 °.

After electrophoresis on a 1% agarose gel, we isolated 1.68 thousand b.p. size BamHI-BglII fragment and ligated with 3mg of pIC19R plasmid digested with BglII in ligation buffer

After ligation, DNA was transformed into competent E. coli HB101 stem cells and incubated for 1 hour. 0 ° C and 5 min. At 42 ° C. The cell mixture was then diluted together in LB medium (1¾ tryptone, 0.5% yeast extract, 1¾ NaCl) and incubated at 37 ° C for 1 h, then plated on agarized LB medium with ampicillin (100 mcg / ml). Among the transformants, clones containing the plasmid pIC19RS were selected by restriction analysis. 50mg of pIC19RS plasmid was digested with Eco147I (100 units) for 2 hours. At 37 ° C, 200 mL of buffer B. The resulting reaction mixture was treated with phenol-chloroform 1: 1, centrifuged, DNA-ethanol-ethanol dissolved in 200 mL of ligation buffer with ATP and added 0.1 opt. of a synthetic oligonucleotide from 53 b.p. homologous to the 5 'end of the preS2 gene:

5 'CT AGA CAA TGG AAC TCC ACT TTC CAC CAA ACT TTG CAA

3 ¹ T GTT ACC TTG AGG TGA AAG GTG GTT TGA AAC GTT

GAC CCA AGA GTT AGA GG-3 '

CTG GGT TCT CAA TCT CC ~ 5 '

-9EN 3988 B

Xbal and incubated with gel, isolated by ligation with 3 mcg

We were 16 or so. At 12 ° C. The reaction mixture was ligated for 20 min. heated at 70 ° C. DNA was ethanol transplanted, dissolved in 200 µl of buffer O, added 10 units. restriction enzyme BglII is 200 units. restriction enzymes; At 37 ° C.

This mixture was fractionated with 1% agarose 0.85 thousand b.p. An XbaI-BglII fragment by pP-T plasmids digested with XbaI and BglII.

After ligation, the resulting cells were transformed into E. coli HB101 competent cells and seeded on LB medium with ampicillin. Clones containing the plasmid p37-pS2-1 were selected.

50mg of p37-pS2-1 were dissolved in 200mL of buffer G (10mM trisH. Cl, 10mM MgCl, 50mM NaCl, 1mM DTT; pH 7.5) at 100 units. KpnI and HindIII restriction enzymes and incubated for 2 h. At 37 ° C. The resulting mixture was fractionated on a 1% agarose gel and isolated at 1.5,000 b.p. The KpnI - HindIII fragment was ligated with 3 mkg of pTZ19R plasmid (Pharmacia, Sweden) digested with KpnI - HindIII restriction enzymes.

The resulting mixture was transformed into competent cells of E. coli DH5a strain and seeded on LB medium with ampicillin. Clones containing the p37-pS2-2 plasmid were selected among the transformants (Fig. (Fig.4). 4).

50mg of p37-pS2-2 were dissolved in 200mL of buffer R (10mM trisHCl, 10mM MgCl, 100mM KCl, 1mM DTT; pH 8.5) and added 100 units. EcoRI restriction enzymes were incubated for 2 h. At 37 ° C.

The DNA was ethanol transplanted and dissolved in 200 mL of Klenov enzyme buffer (50 mM tris-HCl, 6 mM MgCl2, 1 mM mercaptoethanol; pH 7.6) to 0.1 mM deoxynucleotide triphosphates, 3 units. Klenov enzyme and incubated for 30 min. At 20 ° C. The reaction mixture was then stirred for 20 min. incubated at 70 ° C, ethanol transplanted with DNA, dissolved in 200 µl buffer R, added 100 units. restriction enzymes are incubated with 2 or. At 37 ° C.

The resulting mixture was fractionated on a 1% agarose gel and isolated

I. 5 thousand b.p. The EcoRI-HindIII fragment, which was ligated with 3mg of the YepSecl plasmid (Baldari et al., EMBO J., 1987, vol.6, p. 229-234), was digested with HindIII and XhoI restriction enzyme and exposed to Klenow enzyme.

-10LT 3988 B

After ligation, the resulting cells were transformed into E. coli HB1O1 competent cells and seeded on LB medium with ampicillin. Clones containing the p6pS2 plasmid were selected.

mkg of p6pS2 plasmid was dissolved in 200 µl of buffer O and added 50 units each. Ncol and Eco91I and incubated for 2 hours. At 37 ° C, then inactivated the enzymes for 20 min. At 70 ° C. DNA was ethanol transplanted, dissolved in 200 ml Klenov enzyme buffer, deoxynucleotide triphosphate, 3 units. The Klenov enzyme was incubated for 1 h. At 20 ° C. The resulting mixture was fractionated on an agarose gel to give 4.3 thousand b.p. The NcoI-Eco91I fragment was ligated with 3 mkg of pRA1 plasmid digested with SmaI restriction enzyme.

After ligation, the resulting cells were transformed into E. coli HB101 competent cells and seeded on LB medium with ampicillin. Among the resulting clones, plasmid containing pFpS2-48 was selected (Fig. (Fig.5). 5).

We analyze the plasmid as follows:

1. The presence of an ampicillin resistance gene is demonstrated by the ability of the E.coli strain harboring plasmid pFpS2-48 to grow in agarized media containing 50 to 100 mcg / ml ampicillin.

2. The presence of the FOR-R gene for resistance to formaldehyde is demonstrated by the ability of the S.cerevisiae strain containing the FOR-R gene plasmid to grow in agarized medium containing 3-10 mM formaldehyde.

3. The ability of plasmid pFpS2-48 to encode the synthesis of human hepatitis B virus surface antigen HBpreS2Ag is demonstrated by transformation of this strain with S.cerevisiae FH4C strain, selection of formaldehyde-resistant transformants, followed by radioimmunoassay of the resulting transformants. Data are given in the description of this strain.

4. Cleavage of plasmid pFpS2-48 by EcoRI restriction enzyme yields 1.0,

4.8 and 4.9 thousand b.p. size fragments. By splitting BamHI and

BglII restriction enzymes - 2.1 and 8.7 thousand b.p. size fragments.

Cleavage with PstI restriction enzyme - 0.5, 2.2 and 8.1 thousand b.p. size fragments.

-11EN 3988 Example B. Construction of plasmid pFS19.

The mkg p37pS2-2 plasmid obtained as described in Example 4 was dissolved in 200 µl of buffer R and added 100 µl. Hin6I restriction enzyme, incubated at 2 or 37 ° C.

DNA was ethanol transplanted, dissolved in 200 mL Klenov enzyme buffer, deoxynucleotide triphosphate, 3 units. Klenow enzyme was incubated for 30 min at 20 ° C followed by inactivation for 20 min. 70 ° C.

DNA was ethanol transplanted, dissolved in 200 ml buffer 0, added 100 units. BglII restriction enzymes and incubated for 2 h.

At 37 ° C.

After electrophoresis on a 1% agarose gel, we isolated 0.68 thousand. b.p. size fragment, which was ligated with 3 mkg of p6pS2 plasmid sequentially treated with XbaI restriction enzyme, Mung Bean nuclease and BglII restriction enzyme. The newly obtained plasmid was named p5-6S.

Mkg p6-6S was dissolved in 200 µl buffer Y (33 mM tris-acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.5 mM DTT; pH 7.9) and added 100 units each. Eco147I is Eco91I, incubated at 2 or. At 37 ° C.

The DNA was precipitated with ethanol and dissolved in 200 mL of Klenov enzyme buffer, added with 3 units of nucleotide triphosphate.

Klenov enzyme and incubated for 30 min. At 20 ° C.

After electrophoresis, we isolated an 3.9 thousand bp Eco147IEco91I fragment and ligated it with 3 mcg of pRA1 plasmid digested with SmaI restriction enzyme.

After ligation, the resulting cells were transformed into E. coli HB101 competent cells and seeded on LB medium with ampicillin. Clones containing the pFS19 plasmid were selected among the transformants (Fig.6).

Plasmid analysis is performed as described in Example 4.

Cleavage of pFS19 with EcoRI results in 4.9 and 5.5 thousand b.p. size fragments. 1.8 and 8.6 thousand bp by digestion with BglII and BamHI, and by PstI-0.5 digestion,

2.2 and 7.7 thousand. b.p.

example. Construction of the pJLFdSI plasmid.

mkg of pHB 320 plasmid was dissolved in 200 µl of buffer G, added 100 units of BamHI and XbaI and incubated with culture. at 37 ° C.

-12EN 3988 B

After electrophoresis on an agarose gel, we isolated 0.53 thousand b.p. size fragment containing the preS1 and preS2 regions of viral DNA and ligated with 3 mkg of pTZ19R plasmid digested with BamHI and XbaI.

The resulting mixture was transformed into competent HB101 E.coli cells. Among the transformants, we selected clones containing the pTZS1 plasmid.

0.001 mcg of pTZSl plasmid was dissolved in 100 mcg of amplification buffer (67 mM tris-HCl, 6.7 mM MgCl, 1 mM mercaptoethanol, 50 mM KCl, 0.17 mg / ml bovine serum albumin; pH 8.8 at 25 ° C) and added to 0.2 mM concentration. , 5 pcs. Taql DNA polymerase and 0.01 opt. of synthetic oligonucleotides 5 '-ATGGAGAACATCACATCAG3' and 5 '- GCCACCAGCAGGGAAATAC-3'.

The resulting mixture was subjected to 35 cycles of amplification (1 min at 95 ° C, 2 min at 50 ° C, 3 min at 72 ° C). Obtained after polymerase chain reaction 3.4 thousand b.p. size fragment was treated with phenol-chloroform mixture and ethanol transplanted.

DNA was dissolved in 200 ml of ligation buffer, and ATP was added. T4 phage polynucleotide kinases and 50 units. T4 phage DNA ligases. The resulting mixture was incubated for 1 h. at 37 ° C. The resulting mixture was transformed with HB101 E.coli and selected for the plasmid containing the pTZdSI.

mkg pTZdSI plasmid was dissolved in 200 mkl buffer R and added 100 units each. BamHI and EcoRI and incubated at 2 or 37 ° C. The resulting mixture was treated with a phenol-chloroform mixture and the DNA was ethanol-transplanted.

mkg of pTZdSI plasmid digested with BamHI and EcoRI digested with 100 µl of ligation buffer, added ATP, 0.01 opt. pcs. of a synthetic DNA fragment containing the 5 'part of the preSl gene:

5 '-AA TTC TCT AGA GGT CAA AAC TTA TCT ACC TCT AAC CCA

3 '- G AGT TCT CCA GTT TTG AAT AGA TGG AGA TTG GGT

TTA GGT TTC TTC CCA CAC CAC CAA TTG -3 '

AAT CCA AAG AAG GGT GTG GTG GTT AAC CTAG - 5 'pcs. We incubated the T4 phage DNA ligase for 16 h. 12 ° C. The resulting mixture was transformed with HB101 E.coli and selected clones containing the pdS1s plasmid.

-13LT 3988 B mkg pdS1 s plasmid was dissolved in 200 mkl of buffer Y, added 100 units. XbaI restriction enzymes and incubated at 2 or 37 ° C.

After electrophoresis on a 1% agarose gel, 0.49 thousand b.p. size of the Xbal-Xbal DNA fragment and ligated with 8.9 thousand b.p. size fragment derived from p6-pS2 plasmid digested with Xba I restriction enzyme.

The resulting mixture was transformed with HB101 E, coli and selected among the transformants containing the plasmid pJLdSI.

mkg pJLdSI plasmid was dissolved in 200 mkl buffer 0, added 100 units each. Bsp68I and Eco91I and incubated for 2 hours at 37 ° C. DNA was ethanol-transplanted, dissolved in 200 ml Klenov enzyme buffer, added NTP, 3 units. Klenov enzyme and incubated for 30 min. At 20 ° C. DNA was ethanol transplanted, dissolved in 100 ml of ATP ligation buffer, and added 50 units. T4 phage DNA ligases were ligated to 2.2 thousand b.p. size Xbal-PstI pRAI plasmid fragment treated with Mung Bean nuclease. The resulting mixture was transformed with HB101 Ecoli and selected among the transformants containing the plasmid pJLFdS1 (Fig.7).

The plasmid pJLFdSl was analyzed as in Example 4.

The presence of the URA3 marker gene is demonstrated by the ability of this plasmid to compensate for the ura3 mutation.

Cleavage of the plasmid pJLFdS1 with EcoRI results in 3.8 and 7.6 thousand b.p. size DNA fragments. Cleavage with BglII and BamHI results in 1.0 and 10.4 thousand b.p. size DNA fragments. Cleavage with HindIII restriction enzymes 0.25, 0.35, 2.0, and 8.8 thousand b.p. size fragments.

To increase the production of HBsAg, HBpreS2Ag antigenic proteins, a large number of wild-type

S.cerevisiae strains. Fast-growing, polyploidy strains with reduced proteolytic activity were found to have the best productivity (Table 1). The FH4C strain was selected for further work.

-14EN 3988 Table B

S.cerevisiae yeast strains (pFS19) HBsAg content (pieces) 1 * Proteolytic activity (contract units) 2 * DBY746 (D.BotStein) 1 .0 2.75 DC5 (J.Hicks) 1 .5 2.60 a3-4l pep4-l (E.Jones) 4.2 1.35 FH4C (O.Lampen) 10.0 1 .0 VIL-2N2 (App. Inst.) I 3.0 2.5 7A (12) 2.5 2.8 7A (16) 322 2.7 2.8 15B ΓΙ 4 2.3 2.7 442 K2 10.0 1 .2 P6 4.2 2.0 P67 K252 3.8 2.7

1 * determined by radioimmunoassay

2 * determined using HBsAg-labeled I ¹²⁵ sample substrate. Production of S.cerevisiae FH4C [pFS19] (ΒΚΠΜ Y1831) strain and determination of its productivity.

Plasmid pFS19 was transformed with the S.cerevisiae FH4C strain using the Ito method (Ito et al., J. Bacteriol. 1983, T 153, p.163-168).

FH4C [pFS19] stem cells were grown at 30 ° C in 100 ml YEPD medium for 24 h. on a shaker at 200 rpm. After 24 or. we added 4¾ galactose and continued to grow the cells for 8-12 hours. in analogous conditions. After 8-12 or. samples were taken and analyzed.

ml of cell culture was centrifuged for 10 min. At 3000 rpm, cells were resuspended in 1 mL of buffer (0.05 M Na-phosphate buffer, 0.05% Triton X-10Q, pH 7.2), added with 1 g of 0.5 mM glass pellet, and disrupted for 4 min, followed by periodic freezing in ice, followed by centrifugation. 10 min 3000 rpm

Human hepatitis B virus surface antigen HBsAg was quantified by radioimmunoassay using kits for HBsAg (Radiopreparat, Η3Φ YaCCP)

Standard preparations from the Institute of Medical Control were used to obtain a calibration curve. The data are shown in Table 2.

8.9 examples. S.cerevisiae FH4C [pFpS2-48] (ΒΚΠΜ Y1833) and S. cerevisiae FH4C [pJLFdS1] (ΒΚΠΜ Y1832) were obtained and verified in analogy to Example 7, only the plasmids pFpS2-48 and pJLFdSI were used for transformation. The data are shown in Table 2.

table

Expression level of surface antigenic proteins HBsAg, HBpreSIAg and HBpreS2Ag in yeast (% indicated represents the amount of immunologically active protein relative to total soluble protein).

The system HBpreSIAg HBpreS2Ag HBsAg S. cerevisiae according to EP 0164556 0.2% S. cerevisiae EP 0174444 0.18% 0.067% S. cerevisiae EP 0248410 HBpreSIAg + HBpreS2Ag 0.36 mcg / ml 0.69 mg / ml P. pastoris EP 0339567 9-60 mcg / ml 0.11-0.63% P. pastoris EP 0339567 HBpreS2 + HBsAg 1% S. cerevisiae EP 0401941 2.4 mg / ml Claimed system 0.5-0.8% 50-90 mg / ml 0.7-1.3% 80-160 mcg / ml 0.7-1.3% 80-160 mcg / ml

It can be seen from Table 2 that the claimed group according to the yield of human hepatitis B virus surface antigens as a percentage of the total cellular protein exceeds the known analogues in S. cerevisiae yeast by up to 20 times and by yield from 1 l of culture up to 100 times. According to the efficiency of antigen synthesis, the given system is slightly higher than the most efficient system of the currently known Pichia pastoris. S. cerevisiae yeast is convenient because it has long been used in the food industry and is free of toxins and pyrogenic substances.

-16EN 3988 B

High antigen yield in the claimed system is achieved by utilizing an inducible hybrid GALI-10-PYK1 promoter, a PGK1 gene transcription terminator, and a dominant gene FOR-R allowing selection of wild-type S. cerevisiae strains with increased antigen productivity. The use of the dominant gene allows the use of wild-type strains, which can be cultivated using complete, low-cost media, an important advantage in industrial yeast cultivation.

The use of complete media increases the yield of biomass from the same volume of culture fluid, and also shortens the fermentation time by shortening the yeast cell generation time.

The use of the yeast FH4C strain with reduced proteolytic activity reduces the degradation of heterologous proteins, thus facilitating the purification of native hepatitis B virus antigens. The use of the dominant gene allows selection of strains with increased heterologous protein productivity.

Claims (10)

DEFINITION OF INVENTION 1. A recombinant plasmid pFS19 encoding human hepatitis B virus surface S antigen synthesis is 10.4kbp size and consists of the following elements: Kpnl-Xbal 2.9kb.p. a pTZ19R-sized plasmid fragment containing a gene conferring ampicillin resistance and an E. coli ori region; XbaI-BamHI 3.6kb.p. a C. maltosa DNA fragment containing a FOR-R gene that confers formaldehyde resistance (dominant gene); Eco1471-HindIII 2.16 thousand b.p. a size fragment containing a GAL 1-10 transcriptional activator, a pyruvate kinase PYK1 gene promoter, a human hepatitis B virus S gene, a phosphoglycerate kinase PGK1 gene transcription terminator; HindIII-Eco91I 1.74 thousand b.p. size yeast 2 mkm plasmid fragment containing the ori region. 2. The recombinant plasmid pFpS2-48 encoding human hepatitis B surface preS2 antigen is 10.8kbp size and consists of the following elements: Kpnl-Xbal 2.9kb.p. a pTZ19R-sized plasmid fragment containing a gene conferring ampicillin resistance and an E. coli ori region; XbaI-BamHI 3.6kb.p. a C. maltosa DNA fragment containing a FOR-R gene that confers formaldehyde resistance (dominant gene); Ncol-HindlII 2.56 thousand b.p. a size fragment containing a GAL 1-10 transcriptional activator, a pyruvate kinase PYK1 gene promoter, a human hepatitis B virus preS2 gene, a phosphoglycerate kinase PGK1 gene transcription terminator; HindIII-Eco91I 1.74 thousand b.p. size of a yeast 2 mkm plasmid fragment containing the yeast ori region. 3. The recombinant plasmid pJLFdSl encoding human hepatitis B surface preS1 antigen is 11.4kbp size and consists of the following elements: Xbal-Pstl 2.2kb.p. a size C.maltosa DNA fragment containing a FOR-R gene that determines formaldehyde resistance (dominant gene); HindIII-Eco91I 1.74 thousand b.p. yeast 2 mkm plasmid fragment containing the yeast ori region; HindlII-EcoRI 1.74 thousand b.p. a size fragment containing a pyruvate kinase PYK1 gene promoter, human hepatitis B virus preS1 gene, a phosphoglycerate kinase PGK1 gene transcription terminator; EcoRl-Bsp68l 5.63kb.p. size fragment containing the GAL 1-10 transcriptional activator, the yeast URA3 gene, the ampicillin resistance gene, and the E. coli ori region. 4. A method of constructing plasmids pFS19, pFpS2-48 and pJLFdSI encoding the synthesis of surface antigens HBsAg, HBpreS2Ag and HBpreSIAg, which comprises fusing the dominant gene FOR-R in a single plasmid with the S, preS2 or preS1 genes regulated by hybrids. galactose-inducible GAL1-10-PYK1 promoter and PGK1 transcription terminator, as well as a yeast 2mkm plasmid fragment containing the yeast ori region and the ampicillin resistance and E.coli ori regions, whereas the cloning of the dominant gene FOR-R includes the Candida maltosa gene construction of a bank using a bifunctional yeast vector, transformation into Saccharomyces cerevisiae, selection of transformants complementing the leu2 mutation, selection of transformants that can grow on formaldehyde media, isolation of plasmids from these transformants, subcloning of the FOR-R gene and sequencing thereof. 5. The method of claim 4, wherein the plasmid pFpS2-48 constructs plasmid pHB320 by digestion with DraI, ligation with BglII linkers, digestion with BglII and BamHI isolated at 1.68,000 b.p. size fragment is ligated with pIC19R plasmid digested with BglII, the resulting pIC19RS plasmid is digested with Eco147I and ligated to a synthetic oligonucleotide 5 'CT AGA CAA TGG AAC TCC ACT TTC CAC CAA ACT TTG CAA 3' T GTT ACC TTG AGG TGA AAG GTG GTT TGA AAC GTT GAC CCA AGA GTT AGA GG-3 ' CTG GGT TCT CAA TCT CC-5 'followed by digestion with Xbal and BglII isolated at 0.85,000 b.p. size fragment is ligated with pP-T plasmid digested with XbaI and BglII, digested with ppI-pS2-1 and isolated with a PYKI gene promoter, preS2 gene, and a PGK1 terminator cloned into pTI19R plasmid pTI19R. and Hind III restriction enzymes; the resulting plasmid p37pS2-2 is digested with EcoRl, digested with Klenov and digested with HindIII and the resulting 1.51,000 b.p. size fragment is ligated with the YepSecl plasmid fragment obtained by sequential treatment with XhoI restriction enzyme, Klenov enzyme and HindIII restriction enzyme, followed by cleavage of the resulting p6pS2 plasmid with Ncol and Eco91I restriction enzymes, yielding 4.3 thousand b.p. size of the DNA fragment and ligated with pRA1 plasmid digested with SmaI restriction enzyme. 6. The method of claim 4, wherein the plasmid pFS19 is constructed by cleaving plasmid pHB320 with DraI restriction enzyme, ligating with BglII linkers, cleaving with BglII and BamHI restriction enzymes, isolated at 1.68,000 b.p. size fragment is ligated with pIC19R plasmid digested with BglII, the resulting pIC19RS plasmid is digested with Eco147I and ligated to a synthetic oligonucleotide 5 'CT AGA CAA TGG AAC TCC ACT TTC CAC CAA ACT TTG CAA 3' T GTT ACC TTG AGG TGA AAG GTG GTT TGA AAC GTT GAC CCA AGA GTT AGA GG-3 ' CTG GGT TCT CAA TCT CC -5 ′, followed by cleavage with Xbal and BglII, isolated at 0.85 thousand b.p. size fragment is ligated with pP-T plasmid digested with XbaI and BglII, digested with pp-I1 and HindIII, and isolated fragment containing the PYK1 gene promoter, preS2 gene and PGK1 terminator cloned into pTZ19R plasmid. and HindIII restriction enzymes; the resulting p37pS2-2 plasmid is digested with EcoRI, Klenow enzyme digested with HindIII and 1.51,000 b.p. size fragment is ligated with the YepSecl plasmid fragment obtained by sequential treatment with XhoI restriction enzyme, Klenow enzyme and HindIII restriction enzyme, followed by ligation with the resulting p6pS2 plasmid, XbaI restriction site, Mung Bean nuclease and BglII restriction enzyme, with 0.68 thousand bp. a fragment isolated from plasmid p37pS2-2 by treatment with Hin6I restriction enzyme, Klenow enzyme and BglII restriction enzyme; The resulting plasmid p6-6S is cleaved with Eco147I, Eco91I, Klenov enzyme, and isolated 3.9,000 b.p. size fragment, which is ligated with pRA1 plasmid digested with SmaI restriction enzyme. 7. The method of claim 4, wherein the plasmid pJLFdSI is cleaved with BamHI and XbaI restriction enzymes isolated at 0.53 thousand b.p to construct plasmid pJLFdSI. size fragment is ligated with the plasmid pTZ19R digested with BamHI and XbaI, the resulting plasmid pTZS1 is used in place of the matrix in a polymerase chain reaction using 2 synthetic nucleotides as primers in this reaction. 5 '- ATGGAGAACATCACATCAG - 3' and 5 '- GCCACCAGCAGGGAAATAC - The sixth product of 3.4kb DNA is affected by polynucleotide kinase and ligase, the resulting pTZdSI plasmid is cleaved with BamHI and EcoRI, and digested with 5' , 5'-AA TTC TCT AGA GGT CAA AAC TTA TCT ACC TCT AAC CCA 3'- G AGT TCT CCA GTT TTG AAT AGA TGG AGA TTG GGT TTA GGT TTC TTC CCA CAC CAC CAA TTG -3 ' AAT CCA AAG AAG GGT GTG GTG GTT AAC CTAG-5 ', the resulting pdS1s plasmid is cleaved with Xba I restriction enzyme, 0.49 thousand b.p. size of the Xbal-Xbal fragment ligated with 8.9 thousand b.p. a size DNA fragment obtained by digestion of p6pS2 with Xba I restriction enzyme; the resulting plasmid pJLdSI is cleaved with Bsp68I and Eco91I, acts with the Klenov enzyme and ligated to 2.2 thousand b.p. size of the Xbal-Pstl pRA1 plasmid fragment affected by Mung Bean nuclease. 8. Strain Saccharomyces cerevisiae FH4C [pFS19] (ΒΚΠΜ Y1831) is a producer of human hepatitis B virus surface antigen. 9. Saccharomyces cerevisiae strain FH4C [pFpS2-48] (ΒΚΠΜ Y1833) is a producer of preS2 antigen on human hepatitis B virus surface. 10. Saccharomyces cerevisiae strain FH4C [pJLFdSl] (ΒΚΠΜ Y1832) is a producer of preS1 antigen on human hepatitis B virus surface.