RU2115730C1 - Recombinant plasmid pfs 19 determining synthesis of the surface antigen s of human virus hepatitis b - Google Patents

Abstract

FIELD: virology, molecular biology, genetic engineering. SUBSTANCE: method involves construction of recombinant plasmid pFS 19 combining gene determining resistance to formaldehyde FOR-R as a dominant selective marker and gene S encoding the surface antigen of virus hepatitis B expression of that is regulated by hybrid promoter GAL 1-10-PYKI and transcription terminator PGK1 with DNA fragment 2-mcm of yeast plasmid containing ori and with gene that determines resistance to ampicillin with E. coli ori. At transformation of yeast Saccharomyces cerevisiae with obtained plasmid expression level of the surface antigen is 50-160 mg/1 cultural fluid. Invention provides preparing the surface antigen of virus hepatitis B in yeast S. cerevisiae. EFFECT: increased yield of antigen. 2 tbl, 7 dwg

Description

 The invention relates to the field of biotechnology and relates to a method for producing hepatitis B virus antigens in Saccharomyces cerevisiae yeast. Human hepatitis B virus surface antigens can be used to create highly effective recombinant vaccines and diagnostics.

 Many of the known methods for producing human hepatitis B virus surface antigens are based on the use of recombinant yeast strains. The use of yeast is due to the fact that they assemble 22-micron particles consisting of an immunologically active protein. These particles are similar to the 22-micron particles found in the plasma of people who carry hepatitis B virus (HBV).

 An increase in the synthesis of human hepatitis B virus surface antigens in yeast is achieved through the use of recombinant plasmids with more efficient regulatory elements (regulatory promoters, transcription terminators), and also depends on the choice of the plasmid recipient strain.

 It is known to use a number of recombinant yeast plasmids that determine the synthesis of surface antigens HBsAg6 HBpreSlAg and HBpreS2Ag5.

Pyk1-6, GAP1-6 et al., EP 0164556, class. C 12 N 15/00, 1985;
pHBSGAP 347 / 33T and pHBpreSGAP 347 / 33T, EP 0174444, 1986;
pYGAP / pSSA, pYGAP / PSSC, pYGAL / PSSA6 pYGAL / PSSC, pYADH2 / PSSA and pYADH / PSSC, EP 0244924, 1987;
pGG6, pGG61 / Tn-1 and pGG63, EP 0248410, 1987;
pHBpreSGAP 347 / 19T and pYGpreS2S1, EP 0312159, 1989;
pTB05A and pHB6, EP 0339567, 1989; and EP 341746, 1989;
pPHO P31-R, pPHO P31-W, pGLD P31-R and pPKT P31-R, EP 0 401 941, 1990;
and
pYeHBS, GB 2104902, 1983; pRIT10673 and pRIT10674, AU-B-16750 / 83.1983;
RAN201, RAN203 and pAH205, SU 1387878, 1988.

 The disadvantage of using these plasmids is the low level of biosynthesis of surface HBV antigens.

 The closest analogue of the invention is described in the application for European patent 0164556, cl. C 12 N 15/00, 1985, and relates to plasmid PYK 5 containing the auxotrophic yeast marker leu2 ori 2-μm plasmid of the yeast Saccharomyces cerevisiae, the ampicillin resistance gene, human hepatitis B virus surface protein S gene, the expression of which is regulated by hybrid GAL 1 -10-PYK-promoter and terminator of transcription of ADH1.

 The main drawback of this hepatitis B virus surface protein expression system in saccharomycetes yeast (in particular, S. calstergensis 2150-2-3) is the need to use strains mutant for the leu2 gene and, therefore, minimal synthetic media to maintain the recombinant plasmid PYK 5 in the corresponding strain. The strains that produce surface antigens of hepatitis B virus in such media grow more slowly, and the yield of cell biomass and target protein is low. The maximum yield of the immunologically active HBsAg protein using the PYK 5 plasmid is 1.4 mg / g of soluble cell protein, and the maximum yield in this system using other promoters reaches 2.1 mg / g.

 The technical result of the invention is to increase the yield of surface antigen (HBsAg) of human hepatitis B virus in S. cerevisiae yeast.

 The implementation of the technical result is achieved using a new recombinant plasmid pFS19, which determines the synthesis of surface antigen HBV and a new strain of Saccharomyces cerevisiae FH4C / pFS19, which provides an expression level of 40 - 80 mg of protein of surface antigen per 1 liter of culture (0.4 - 0.8% immunologically active protein with respect to all soluble cellular protein).

Recombinant plasmid pFS19, which determines the synthesis of hepatitis B virus surface antigen, is characterized by the following features:
has a size of 10.4, etc.,
consists of:
Kpn 1-Xba I, fragment of plasmid pTZ19R, 2.9 kbp, containing the gene for resistance to ampicillin and ori E. coli;
Xba I-BamH 1 C. maltosa DNA fragment of 3.6 kbp containing the FOR-R formaldehyde resistance gene (dominant selectable marker);
An Eco147 I-Hind III fragment of 2.16 kbp containing a GAL 1-10 transcription activator, a pyruvate kinase gene promoter PYK I, a human HBV surface antigen S gene, a PGK 1 phosphoglycerate kinase transcription terminator;
Hind III-Eco91 1 DNA fragment of 2 μm yeast plasmid of 1.74 bp in value containing ori;
has unique restriction enzyme recognition sites DamH I, Bgl II, Sal I.

 Figure 1 shows the scheme for cloning the formaldehyde resistance gene FOR-R.

 In figure 2, 3 is the nucleotide sequence of the FOR-R gene.

 In FIG. 4 is a schematic of the preparation of a pRT plasmid containing the PYK I promoter and the PGK 1 gene transcription terminator.

 Figure 5 - scheme for the preparation of plasmid p37pS2-2.

 In Fig.6 scheme for obtaining plasmids p6pS2.

 Figure 7 - scheme for obtaining plasmid pFS19.

 The basic principle of constructing the pFS19 plasmid is to combine the FOR-R formaldehyde resistance gene into a single plasmid as the dominant selectable marker, the S gene, the expression of which is regulated by the GAL1-10 - PYK I hybrid galactose-induced promoter and PGK 1 transcription terminator, 2 μm DNA fragment yeast plasmids containing ori, ampicillin resistance gene and E. coli ori. Moreover, the cloning of the dominant FOR-R selective marker includes obtaining a Candida maltosa gene library in a bifunctional yeast vector, subsequent transformation of Saccharomyces cerevisiae and selection by the auxotrophic marker leu 2; selection of transformants capable of growing on a medium with formaldehyde; isolation of plasmids from formaldehyde resistant transformants and containing the FOR-R gene; subcloning and sequencing of the FOR-R gene.

 The main steps in the construction of a surface antigen producer are as follows.

 1. Cloning of a dominant selective marker of FOR-R formaldehyde resistance gene.

 2. Cloning of the PGK 1 (phosphoglycerate kinase) and PYK I (pyruvate kinase) genes.

 3. Construction of an expression cassette consisting of the PYK I promoter and PGK 1 transcription terminator.

 4. Construction of a plasmid expressing the surface antigen of hepatitis B virus.

 5. Search for strains of Saccharomyces cerevisiae with reduced proteolytic activity and overproduction of human hepatitis B virus surface antigen.

 The preparation of a new strain producing a surface virus S antigen of human B virus is based on transformation of the wild-type S. cerevisiae polyploid yeast strain plasmid pFS19 with reduced proteolytic activity according to the FOR-R marker and subsequent selection of the best producer of human surface HBV S antigen.

 The strain Saccharomyces cerevisiae FH4C / pGS19 VKPM U 1831, producing surface antigen S HBV, is characterized by the following features.

 Morphological signs. The cells are oval.

 Cultural signs. Cells grow well on YEPD (2% peptone, 2% glucose, 1% yeast extract, 2% agar). When growing on solid media - smooth, white colonies, which during storage become rough. They grow well in liquid YEPD media (2% peptone, 2% glucose, 1% yeast extract).

Physiological and biological signs. Cells grow at a temperature of from 10 to 37 ^o C at an optimum pH of from 5.0 to 6.0.

 As a carbon source, the strain assimilates glucose, galactose.

 As a source of nitrogen - ammonium salts, peptone, tryptone.

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

 It is resistant to formaldehyde up to 10 mm.

 Proteolytic activity is reduced.

 Example 1

Cloning of the dominant selective marker FOR-R
Candida maltosa yeast is grown to the late logarithmic phase in YEPD medium (1% yeast extract, 2% glucose) and chromosomal DNA is isolated from the cells. Chromosomal DNA is partially digested with restriction enzyme Sau3A and the resulting fragments are ligated with plasmid pL3, digested with restriction enzyme BamHI and treated with alkaline phosphatase. Ligation DNA is used to transform competent cells of E. coli strain HB101. The total recombinant DNA was isolated from E. coli cells and S.cerevisiae DBY736 (his3 leu2 trp1 ura3) was transformed with the resulting C. maltosa gene library. After transformation, cells are plated on minimal medium containing 50 μg / ml of histidine, uracil and tryptophan. Transformants selected by the Leu ⁺ phenotype by the replica method were transferred to complete YEPD medium with 3-10 mM formaldehyde. Transformants growing on a medium with 3-10 mM formaldehyde are selected. When analyzing plasmids isolated from yeast transformants and transformed into E. coli, it is found that all plasmids contain a common portion of Candida maltosa DNA. By further subcloning, an Xbal-BamHI C. maltosa DNA fragment of 3.6 kb was isolated. encoding the FOR-R formaldehyde resistance gene (plasmid pRA1, FIG. 1). The Xbal-BamHI fragment is sequenced and one long open reading frame containing an intron and a coding protein of 380 amino acids is detected (Fig. 2, 3). The FOR-R gene cloned on multi-copy plasmids containing ori 2 μm DNA determines resistance to formaldehyde in concentrations of 3-10 mM and allows the transformation of wild-type S. cerevisiae strains.

 Example 2

Cloning of PGK 1 and PYK I genes
To clone the PYK I (pyruvate kinase) and PGK 1 (phosphoglycerate kinase) genes with the S.cerevisiae gene library based on the pL3 plasmid, S.cerevisiae DFY331 (leu2 pgkI) and Pyk 1-5 (adeI leui metI4 ura 3 py 5) and plated on synthetic medium without leucine containing glucose as a carbon source. Strains mutant for the genes PYK I and PGK 1 are not able to grow on a medium with glucose. Plasmid DNAs are isolated from the grown transformants and, with further subcloning, the PYK I and PGK 1 genes are isolated (Fig. 3 of the plasmid pL3-PYK and pL3-PGK).

 Example 3

Construction of an expression cassette consisting of a PYK I promoter and PGK 1 transcription terminator
The cloned genes PGK 1 and PYK I are used as a source of regulatory elements for constructing the expression vector. Plasmid pL3-PGK was digested with restriction enzymes BglII and HindIII. An isolated 0.38 kb BglII-HindIII fragment containing the PGK 1 gene transcription terminator is ligated with plasmid pIC19R (March et al., Gene, 1984, T. 32, p. 481 - 485), digested with restriction enzymes BglII and HindIII. The resulting plasmid is designated pPGKT. Plasmid pL3-PYK was digested with restriction enzymes EcoRI and BglII, a 1.0 kbp fragment was isolated containing the PYK I gene promoter and part of the coding sequence, and ligated with plasmid pPGKT digested with restriction enzymes EcoRI and BglII. The resulting plasmid is designated pRT (FIG. 4). The plasmid pRT contains an expression cassette consisting of a promoter of the PYK I gene (EcoRI-XbaI) and a PGK 1 gene transcription terminator (BglII-HindIII).

 Example 4

Construction of plasmid pFS19
50 μg of the plasmid pHB320 (Punpen et al. DAH USSR, 1983, v. 271, pp. 230 - 235) was digested with restriction enzyme DraI (100 units) in 200 μl of buffer B (10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM DTT; pH 7.5) for 2 hours at 37 ^° C, after which the restriction enzyme is inactivated by heating at 70 ^° C for 20 minutes, the DNA is precipitated by adding 20 volumes of ethanol, centrifuged for 3 minutes at 1500 rpm, then the DNA is dissolved in 200 μl of ligation buffer (50 mM Tris-HCl, 10 mM MgCI, I mM DTT; pH 7.5), adenosine triphosphate (ATP), 0.01 optical units (OE) of BglII linkers and 50 units are added. T4 ligase DNA; ligated for 16 hours at 12 ^o C.

The resulting mixture was precipitated with ethanol, dissolved in 200 μl of buffer O (50 mM Tris-HCI, 10 mM MgCI, 100 mM NaCl, I mM DTT; pH 7.5) and treated with restriction enzymes BamH I (50 units) and Bgl II (100 units) for 2 hours at 37 ^o C.

 After electrophoresis on a 1% agarose gel, a 1.68 bp BamH I-Bgl II fragment was isolated. and ligated with 3 μg of plasmid pIC19R digested with restriction enzyme Bgl II in ligation buffer.

,
и лигируют 16 ч при 12^oC. Смесь после лигирования прогревают 20 мин при 70^oC, ДНК переосаждают этанолом, растворяют в 200 мкл буфера О, добавляют 100 ед. рестриктазы Bgl II и 200 ед. рестриктазы Xba I и инкубируют 2 ч при 37^oC.After ligation, the competent cells of the E. coli strain HB101 are transformed with a DNA mixture, the mixture is incubated for 1 h at 0 ^° C and 5 min at 42 ^° C. After 10-fold dilution with LB broth (1% peptone, 0.5% yeast extract, 1% NaCl), the transformed cells were grown for 1 h at 37 ^° C and plated on LB agar medium with ampicillin (100 μg / L). Among transformants, clones containing plasmid pIC19RS were selected using restriction mapping. 50 μg of plasmid pIC19RS was digested with restriction enzyme Eco147 I (100 units) in 200 μl of buffer B for 2 h at 37 ^° C. The resulting mixture was treated with phenol-chloroform (1: 1), centrifuged, DNA was reprecipitated with ethanol, dissolved in 200 μl buffer for ligation with ATP, add 0.1 opt. 53 bp synthetic oligonucleotide containing the 5'-sequence of the preS2 gene:

,
and ligated for 16 hours at 12 ^° C. After ligation, the mixture is heated for 20 minutes at 70 ^° C, DNA is reprecipitated with ethanol, dissolved in 200 μl of buffer O, 100 units are added. restriction enzymes Bgl II and 200 units. restriction enzymes Xba I and incubated for 2 hours at 37 ^o C.

 After fractionation of this mixture on a 1% agarose gel, an 0.85 kb Xba I-BglII fragment was isolated which was ligated with 3 μg of pP-T plasmid digested with restriction enzymes Xba I and Bgl II.

 The mixture obtained after ligation was transformed into competent cells of E. coli strain HB101 and plated on LB medium with ampicillin. Among clones, plasmid p37-pS2-1 was selected.

50 μg of the plasmid p37-pS2-1 was dissolved in 200 μl of buffer G (10 mm Tris-HCl, 10 mm MgCl, 50 mm NaCl, 1 mm DTT; pH 7.5), add 100 units restriction enzymes Kpn 1 and 100 units. Hind III restriction enzymes and incubated for 2 hours at 37 ^° C. The resulting mixture was fractionated on a 1% agarose gel and a 1.5 kb Kpn I-Hind III fragment was isolated which was ligated with 3 μg of plasmid pTZ19R (Pharmacia, Sweden ) digested with restriction enzymes Kpn 1 and Hind III.

 The resulting mixture was transformed into competent cells of E. coli strain DH 5a and plated on LB medium with ampicillin. Among the transformants, clones carrying the p37-pS2-2 plasmid were selected (FIG. 5).

50 μg of the plasmid p37-pS2-2 was dissolved in 200 μl of buffer R (10 mm Tris-HCl, 10 mm MgCl ₂ 100 mm NaCl, 1 mm DTT; pH 8.5), add 100 units restriction enzymes EcoRI and incubated for 2 hours at 37 ^o C. DNA is reprecipitated with ethanol and dissolved in 200 μl of Klenov fragment buffer (50 mM Tris-HCl, 6 mM MgCl ₂ , 1 mM mercaptoethanol; pH 7.6), deoxynucleoside triphosphates are added to a concentration of 0 , 1 mm, 3 units The DNA polymerase I of the Klenov fragment was incubated for 30 min at 20 ^o C. The mixture was then heated for 20 min at 70 ^o C, the DNA was reprecipitated with ethanol, dissolved in 200 μl of buffer R, 100 units were added restriction enzymes Hind III and incubated for 2 hours at 37 ^o C.

 The resulting mixture was fractionated on an agarose gel and an 1.51 kb EcoRI-HindIII fragment was isolated which was ligated with 3 μg of YepSecl plasmid (Baldari et al., EMBO J., 1987, v. 6, p. 229 - 234) digested with restriction enzymes HindIII and XhoI and treated with a Klenov fragment.

 The resulting mixture transform strain E. coli HB101. Among the transformants, clones containing the plasmid, which is designated p6pS2, are selected.

50 μg of plasmid p37pS2-2 was dissolved in 200 μl of buffer R, 100 units were added. Hind I restriction enzymes, incubated for 2 hours at 7 ^o C.

DNA is reprecipitated with ethanol, dissolved in 200 μl of Klenov fragment buffer, deoxynucleoside triphosphates, 3 units are added. DNA polymerase of the Klenov fragment and incubate the fragment by heating at 70 ^o C for 20 minutes

DNA is reprecipitated with ethanol, dissolved in 200 μl of buffer O, 100 units are added. restriction enzymes BglII and incubated for 2 hours at 37 ^o C.

 After electrophoresis in a 1% agarose gel, a fragment of 0.68 kb is isolated. which is ligated with 3 μg of plasmid p6pS2 sequentially treated with restriction enzyme Xba I, nuclease Mung Bean and restriction enzyme Bgl II; the resulting plasmid is designated p6-6S.

50 μg of plasmid p6-6S was dissolved in 200 μl of Y buffer (33 mM Tris-acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.5 mM DTT; pH 7.9), 100 units were added. restriction enzymes Eco147 I and Eco91 I and incubated for 2 hours at 37 ^o C.

DNA is reprecipitated with ethanol, dissolved in 200 μl of Klenov fragment buffer, nucleoside triphosphates (NTR), 3 units are added. Klenov fragment and incubated for 30 minutes at 20 ^o C.

 After electrophoresis, an Eco147 I-Eco91 I fragment of 3.9 kb is isolated. and ligated with 3 μg of plasmid pRA1 digested with restriction enzyme SmaI. The resulting mixture is transformed competent cells of strain HB101 E. coli and plated on LB medium with ampicillin. Among the transformants selected clones containing the plasmid pFS19 (Fig.6).

 When digesting the plasmid pFS19 with EcoRI restrictase, fragments of 4.9 kb were obtained. and 5.5 kb When digested with restriction enzymes BglII and BamHI, 1.8 kb fragments and 8.6 kbp, and when digested with restriction enzyme Psti, 0.5 kbp, 2.2 kbp and 7.7 kb

 Plasmid analysis is carried out as follows.

 1. The presence of a marker of resistance to ampicillin is confirmed by the resistance of E. coli strains transformed with this plasmid to ampicillin in an agar medium at a concentration of 50-100 μg / ml.

 2. The presence of a formaldehyde resistance marker, FOR-R, is confirmed by the resistance of S. cerevisiae strains transformed with this plasmid to formaldehyde at a concentration of 3-10 mM.

 3. The ability of the pFS19 plasmid to determine the synthesis of the surface hepatitis B virus S antigen in humans is confirmed by testing clones of the S. cerevisiae FH4C strain transformed with this plasmid and resistant to formaldehyde by radioimmunoassay for the production of surface HBV S antigen.

 The research results are given in the description of the corresponding strain.

 To develop systems with increased production of surface antigen HBV (HBsAg), a large number of wild-type recipient strains of S. cerevisiae yeast are tested. It turns out that fast-growing, polyploid strains with reduced proteolytic activity have the best productivity (Table 1). For further work, the FH4C strain is selected.

 Example 5

Obtaining strain S. cerevisiae FH4C / pFS19 VKPM Y1831 and determining its productivity
Plasmid pFS19 transform cells of the strain S. cerevisiae FH4C, as described in (Ito et al., J. Bacteriol., 1983, v. 153, p. 163 - 168).

S. cerevisiae FH4C / pFS19 cells were grown at 30 ^° C in 100 ml of YEPD for 24 hours on a shaker at 200 rpm, galactose was added to a concentration of 4% and the process continued for 8-12 hours. After 8-12 hours, a sample was taken.

 1 ml of cell culture is centrifuged for 10 min at 3000 rpm, suspended in 1 ml of buffer (0.05 M Na-phosphate buffer containing 0.05% Triton X-100, pH 7.2), add 1 g of glass balls with a diameter 0.5 mm, then cooled on ice and centrifuged for 10 minutes at 3000 rpm.

 The content of hepatitis B virus surface antigen S is determined by the radioimmunological method using HBsAg determination kits, and preparations obtained from the Institute of Medical Control (Moscow) as standards for the calibration curve. The data are given in table.2.

 From the table. 2 shows that the claimed system for the yield of the surface hepatitis B virus antigen in humans as a percentage of the total cellular protein surpasses the known analogues by about 20 times, and in yield per 1 liter of culture - by about 100 times. According to the productivity of synthesis, the claimed system is at the same level (only slightly increased), as the most effective P.pastoris system currently known. However, a system based on S. cerevisiae is more developed and familiar to large-scale production cultivation. S.cerevisiae yeast refers to organisms that have passed all medical toxicity tests and are a proven producer of heterologous proteins for medical purposes.

 The increase in antigen yield in the inventive system is achieved through the use of the inducible hybrid GAL 1-10-PYK l promoter, PGK 1 transcription terminator and the dominant FOR-R selective marker, which allows selection of wild-type S. cerevisiae strains with increased antigen productivity. The use of the dominant selective marker FOR-R allows the use of rich, rich, inexpensive media for the cultivation of producers of human hepatitis B antigenic proteins, which is an important advantage in large-scale cultivation.

 When using complete media, the biomass yield is increased based on the same volume of culture fluid, and the fermentation time is also reduced due to a decrease in the generation time of yeast cells.

 Since the FH4C strain has a reduced proteolytic activity, the degradation of heterologous proteins is reduced, which facilitates the purification of native human hepatitis B virus surface antigens. The use of a dominant selective marker makes it easy to screen a large number of wild-type strains in order to select strains with increased productivity of heterologous proteins.

Claims (1)

 Recombinant plasmid pFS 19, which determines the synthesis of human hepatitis B virus surface antigen S, 10.4 kbp, consisting of the following fragments: Kpnl-Xbal fragment of plasmid pT Z19R of 2.9 kbp. containing the gene for resistance to ampicillin and ori Escherichia coli; Xbal-BamHl of a 3.6 kb Candida maltosa DNA fragment containing the FOR-R formaldehyde resistance gene (dominant selectable marker); An Eco147I-HindIII fragment of 2.16 kbp containing a GAL 1-10 transcription activator, a promoter of the pyruvate kinase gene PYKI, a human hepatitis B virus surface antigen S gene, a PGK1 phosphoglycerate kinase gene transcription terminator; HindIII-Eco 911 DNA fragment of a 2 μm yeast plasmid of 1.74 kbp containing the ori sequence.

Images