RU2082759C1 - Strain of yeast saccharomyces cerevisiae containing recombinant plasmid yep 63/ab, - a producer of human hepatitis b virus m-protein derivative - Google Patents

Abstract

FIELD: virology, biotechnology, vaccines. SUBSTANCE: invention relates to preparing the strain-producer of human hepatitis B virus M-protein derivative. Strain has recombinant plasmid YEP 63/AB determining synthesis of hepatitis virus B M-protein derivative in transformed cells. Synthesizing protien is assembled to particles that are similar with natural virus particles with size 22 nm. The level of synthesis of M-protein derivative is 0.5-1% of total cellular protein. New strain-producer can be used for industrial production of human hepatitis B M-protein derivative. Derivative of M-protein can be used for diagnosis and prophylaxis of diseases caused by human hepatitis B virus. EFFECT: new yeast strain. 6 dwg

Description

 The invention relates to biotechnology and medicine, in particular to genetic engineering, and is a recombinant plasmid DNA that determines the synthesis of a derivative of the average surface protein (M protein) of human hepatitis B virus (hepatitis B virus HBV) and a yeast strain containing this plasmid and which is producer of this protein.

 The invention may find application in the production of vaccines against viral hepatitis B and in the diagnosis and prevention of diseases caused by HBV.

 Viral hepatitis B is one of the most common human diseases. According to WHO estimates, more than 300 million people in the world are chronic carriers of this virus. In the USSR, up to 1 million people get viral hepatitis annually. Among them, the proportion of patients with hepatitis B is 9-10. The economic damage is estimated at more than 500 million rubles. annually (Kondrusev, 1990, V: Results of Science and Technology. Virology, vol. 22, 3).

 Existing commercial vaccine preparations created by genetic engineering methods contain the main surface protein of the HBV virus (M-protein) as a protective principle. Such vaccines are not inferior in efficacy to inactivated or subunit vaccines (Ellis Gerety, 1990, J. Med. Virol, 31, 54-58). However, among healthy people, 5 to 15% of recipients do not develop immunity against HBV when immunized with M-protein (Szumness et. Al. 1980, New Engl. J. Med. 303, 833-841). Patients with hemophilia or with impaired immune systems also respond poorly to immunization with such a vaccine. On the other hand, it was shown that re-immunization of such recipients with one of the other two surface proteins of the virus large (L-protein) or medium (M-protein) causes the development of persistent immunity (Zangley et. Al. 1988, 67, 229-245). It is known that the HBV antigens (HBsAg), which are part of the shell of the virion and the so-called 22 nm particles, are represented by the main (S, 226 a.k.o.), average 281 a.k.o.) and large (389 or 400 a.ko.) (depending on the subtype) by surface HBV proteins. These three proteins are the products of one open reading frame (OPC) of the viral genome, divided by three ATG initiator codons into three pre S1, pre S2, and S domains from 5 'to 3'. All of these proteins are capable of collecting in 22-nm lipoprotein particles with the properties of HBsAg and are the subject of intensive practical and theoretical studies in terms of hepatitis B immunoprophylaxis. Numerous immunodominant epitopes have been identified in the pre S regions (Milich et. Al. 1986, Im manol. 137, 2703-2710, Neurath et. Al. 1987, In: Modern Appr. To New Vaccines Including Prevention of AIDS. CSHL, 159). Two such epitopes of the pre S2 region responsible for the T- and B-cell response are mapped at positions 14-24 a.a. on the sequence of the M protein. These epitopes overlap (positions 12–18 aa) the binding site for polymerized human serum albumin (PAR site), which mediates the attachment of the HBV virion to hepatocytes. Apparently, pre S2 glycosylation also plays an important role in this process (Xuanyong Lu et. Al. 1989, In: Hepatitis B Virus CSHL. 97); the pre S2 glycosylation site is located near the main epitopes (positions 4–6 a.c. about. figure 1).

To date, recombinant plasmid DNAs are known that provide expression of the M-protein gene and its derivatives in Sacharomyces cerevisiae yeast (EP 0278940, EP 0312159). It was shown that the recombinant HBV M protein synthesized in S. cerevisiae, carrying a deletion at the Arg ⁴⁸ site, is more resistant to the action of trypsin-like proteases (TPP) of yeast than its natural counterpart (Itoh, Y, Fujisa-wa. Y. 86, Bioch Biophys. Res. Comm. 141, 942-948). At the same time, targets for protease attack are located almost over the entire length of the pre S2 region, although they do not affect the main N-terminal epitopes (Langley at. Al. 1988).

 The aim of the invention is to provide a producer strain of a modified HBV M-protein that is more resistant to yeast CCI compared to the variant obtained by Ito and Fujisawa (Itoh. Y. J Fujisawa, Y. 1986), capable of efficient constitutive synthesis of the target product.

 This goal is achieved by the construction of recombinant plasmid DNA carrying a modified gene of HBV M protein with a deletion of 15 bp in size. in the region responsible for the sensitivity of the pre S2 region to CCI and designated by the authors as pHBmdI. Based on it, the recombinant plasmid DNA YEp63 / AB was constructed that encodes the synthesis of a CCV-resistant derivative of the HBV M protein. The obtained plasmid transformed the yeast strain S. cerevisiae 20B12 [d trpl pep 4-3] J. gas. 1976, Genetics 85, 23-28). The presence in the plasmid of the intact promoter of the yeast gene glyceraldehyde 3-phosphate dehydrogenase (GPD) with an optimized 5'-untranslated region in front of the AUG initiator codon (5'-HTO) allows one to achieve a high level of synthesis of the target product without resorting to induction. The synthesized protein normally collects in a particle like a natural 22 nm HBsAg particle. The synthesis level of the derived M-protein is 1.5-2 of the total protein of the cell.

 The recombinant plasmid pllBmd1 (Fig. 1) contains the replicon and the gene for resistance to ampicillin of the plasmid pUC19, the promoter and part of the LACZ lac gene, the E. coli operon, and the fragment encoding the HBV M protein derivative, which is integrated into the “polylinker” of pUC19 plasmid, of the following elements: EcoRI-BamHI fragment of plasmid pUC19 2642 bp comprising a replication start site, an ampicillin resistance gene (bla), a promoter, and a part of the E. coli lac operon LACZ gene; 1350 bp EcoRI BamHI fragment encoding a derivative of HBV M protein

 The size of the plasmid pHBmd15 is 4017 bp The copy number of the plasmid is about 50 molecules per cell.

 DNA plasmid pHBmd15 contains unique restriction sites EcoRI, BamHI SaIGI, Pst, HindIII, 2 restriction sites XbaI, SphI. The provisions of all the listed restriction sites relative to the unique EcoRI site are shown in table. one.

 The complete primary structure of plasmid pUC19 is known. The primary structure of the fragment encoding the M-protein derivative is shown in FIG.

 The recombinant plasmid YEp63IAB (FIG. 3) contains an expression cassette for the HBV M protein derivative gene integrated into the pJDB 207 yeast shuttle vector, which in turn contains the replicon and plasmid pAT153 ampicillin resistance gene, yeast DNA replicon 2μ, TRPI yeast genes and Teu 2-d. The expression cassette includes the GPD promoter, an optimized 5'-UTO M protein gene, and a phosphoglycerate kinase (PGK) gene transcription terminator.

Plasmid YEp63 / AB consists of the following elements:
Hind III SaIGI ("blunted") fragment of the known plasmid pJOB207 size 6633 BP (JR Broach, Meth, Enzymol. V. 101, p 307-325, 1983);
2134 bp Hind III-Bam III gene expression fragment of a derivative of HBV M protein

 Bam HI-Bg III ("blunt") fragment with the yeast TRPI gene size of 1203 BP from the known plasmid YRp7 (Tsclamper, G Carbon, J, 1980, Gene. 10, 157-166).

Plasmid YEp63 / AB contains the genes:
a modified HBV M-protein gene encoding the synthesis of a derivative of this protein with divisions by amino acid residues from Pro ³⁴ to Asp ⁵² and having an optimized yeast 5 ′ untranslated gene region in front of the ATG codon and an intact transcription terminator sequence.

TRPI gene providing tryptophan prototrophy of transformants of the recipient strain of the yeast carrying the plasmid;
LEU2-d gene providing leucine prototrophy of transformants of a recipient yeast strain carrying a plasmid;
bla-gene, which provides the synthesis of b-lactamase and affects the resistance to ampicillin of E. coli strains carrying the plasmid.

 The size of the plasmid YEp63 / AB is 10070 bp

 Plasmid YEp63 / AB contains unique BamHI, HpaI KpnI restriction sites, 2 PstI restriction sites and 3 XbaI, EcoRI, HinIII restriction sites. The positions of all these sites in restriction relative to a unique site are shown in table. 2.

 The synthesis of the M-protein derivative in yeast is controlled by the yeast GPD promoter. The complete primary structure of all the elements of which the YEp63 / AB plasmid is built is known. The primary structure of the expression cassette, starting from the junction of the promoter with the M-protein gene and ending with the PGK transcription terminator, is shown in FIG. 2.

 Plasmid YEp63 / AB is replicated in E. coli cells due to pAT153 replicon. In yeast cells, plasmid replication is provided by a 2μ replicon of yeast DNA.

The strain producer of the HBV M protein derivative was obtained by transformation of S. cerevisiae 20B12 [d trpl pep4-3] cells with plasmid YEp63 / AB. The synthesized protein normally collects in a particle like a natural 22 nm HBsAg particle. The synthesis level of the derived M-protein according to enzyme-linked immunosorbent assay is 1.6-2 of the total protein of the cell. The mitotic stability of the plasmid YEp63 / AB in the strain S. cerevisiae 20B12 under selective growth is 75-80% of the number of copies of the obtained plasmid per yeast cell from 3 to 4. The strain was deposited in the All-Union Collection of Microorganisms at IBPM AN USSR under VKM number CR-348D . The strain S. cerevisiae 20B12 containing the plasmid YEp63 / AB, characterized by the following features:
Morphological signs: cells have a shape from round to oval, budding when dividing.

 Cultural traits: cells grow well on commonly used culture media. The generation time was about 90 min in YNB liquid medium (0.67% Yeast Nitrogen Base, Difco, 2% glucose). On 2-2.5% nutrient agar "Difco" round, smooth, white colonies with even edges are formed. When grown on liquid YNB and YEPD (1% yeast extract, 2% peptone, 2% glucose), an intense homogeneous suspension is formed.

Physiological and biochemical characteristics: optimal cultivation temperature: from 28 to 30 ^o C, optimum pH 6.0. The carbon source is mainly glucose, in some cases ethanol. A source of nitrogen is organic compounds (peptone, amino acids).

 Example 1

 Construction of recombinant plasmid DNA pHBmdl.

 The design was carried out in accordance with the scheme presented in figure 4.

 To obtain mutations in the HBV M-protein gene that protect the encoded protein from the action of yeast proteases, using the S1 nuclease, the "extra" nucleotides around the XhoI site of the pSMpre S2-S plasmid were removed (Eldarov et al. 1987, Biotechnology, 3 19-26). Hydrolyzed XhoI plasmid DNA was sequentially treated with SI nuclease, Klenov fragment, and T4 phage ligase. The obtained clones were screened by direct sequencing of the isolated plasmids.

10 μg of the plasmid pSMpre S2-S hydrolyzed 2 units XhoI restriction enzymes in 30 μl of the incubation mixture for 2 hours at 37 ^° C in high-salt buffer of the following composition: 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MDCl ₂ , 1 mM DTT. After incubation, 70 μl of distilled water was added and 100 μl of a phenol / chloroform mixture (1 part phenol saturated with Tris-HCl pH 7.5 and 1 part chloroform) was treated by stirring with a shaker (15 s) and centrifuged for 5 min in a benchtop centrifuge "Eppendorf". The upper phase was transferred to a new tube and precipitated with 3 volumes of ethanol in the presence of 0.3 M NaOAc pH 5.0 (freezing at -70 ^° C and centrifuging for 10 minutes at 10 thousand rpm in an Eppendorf benchtop centrifuge). The DNA precipitate was washed with 70% ethanol and, after drying, dissolved in 5 μl of TE buffer (10 mM Tris-HCl, pH 0.1 mM EDTA).

Then 5 μl of 10 x S1 buffer (500 mM NaOAc pH 5.0, 300 mM NACl, 10 mM ZnSO ₄ ), 40 μl bidistilled water and 3 units were added. S1 exonuclease (Amersham). Incubated for 30 min at 30 ^° C, the preparation was extracted with phenol / chloroform, DNA was precipitated with alcohol, dissolved in TE and doped in a volume of 100 μl in a mixture with 10 μl of 10 x ligase buffer (500 mM Tris-HCl pH 7.5, 100 mM DTE, 100 mM MgCl ₂ , 10 mM ATP, 1 mM BSA) and 1 unit. T4 ligases (Amersham) and incubated for 3 h at 15 ^o C. Then the reaction mixture was heated for 10 min at 70 ^o C. Finally, 10 μl of 1 M NaCl and 3 units were added. XhoI (Amersham) and incubated at 37 ^° C. for an hour 200 μl of competent cells of E. coli TGI strain prepared in a standard way (T. Maniatis et al. Molecular cloning. Methods of genetic engineering. M. Mir, 1984), transformed 20 μl of ligase mixture and transformants were selected on nutrient agar plates containing 50 μg / ml ampicillin.

The plasmid DNA from the obtained clones isolated by the standard alkaline method (Maniatis et al. 1984) was analyzed by hydrolysis with restriction enzyme XhoI. Those plasmids that did not contain the XHoI site were analyzed further by sequencing the preS2 region according to the Sanger method (Hattori SaKaKi. 1986, Anal, Biochem. 152, 232-238) using a “universal” primer. As a result, a plasmid encoding an M-protein derivative with a deletion of 18 a.k.o. in the region of Arg ⁴⁸ preS2 region (figure 2) and designated pHBmdI.

 Example 2

Construction of recombinant plasmid DNA YEp63 / AB
The design scheme is presented in figure 5 (A-D).

 A / Construction of plasmid 56 / AB (figure 5).

To create an "expression cassette" of the M protein gene in S. cerevisiae yeast, the gene of the HBV M protein derivative of plasmid pHBmdI was first cloned under the GPD promoter of the GPD / Ba131N2 plasmid at the EcoRI restriction site. For this, 5 μg of DNA of the plasmid pHBmdI was hydrolyzed together with restriction enzymes EcoRI and BamHI (10 units each) in a volume of 25 μl in high salt buffer (Example 1) for 2 h at 37 ^° C. The mixture was fractionated on a 1% agarose gel. A desired DNA fragment of about 1400 bp was eluted from low-melting agarose using a standard procedure (Maniatis et al. 1984). DNA was precipitated with ethanol and dissolved in 5 μl of water.

 To obtain a vector from the plasmid GPD / Ba131N2, 5 μg of DNA was hydrolyzed in 30 μl of the mixture together with restriction enzymes EcoRI and BamHI and a DNA fragment of about 3700 bp from light agarose was eluted. DNA was precipitated with alcohol, dried and dissolved in 5 μl of TE buffer.

0.5 μl of hydrolyzed EcoRI and BamHI GPD / Ba131N2 DNA was mixed with 1 μl of Eco-Bam HI DNA fragment encoding a derivative of the HBV M-protein of plasmid pHBmdI, and mixed with 2 μl of 10x ligase buffer (Example 1). The volume of the solution was adjusted to 20 μl with double-distilled water, 1 unit was added. T4 ligases (Amersham) were incubated for 3 h at 15 ^° C. Then the reaction mixture was heated for 10 min at 70 ^° C. The isolated plasmid DNA from the transformers of E. coli TG1 strain was analyzed with the corresponding restrictase enzymes. As a result, plasmid 56 / AB was selected with the following primary structure of the docking region of the GPD promoter and the M-protein gene:
5'-NTP / GPDI / ⁺ 1 linker ⁺ 1 / preS2 area /
TAACAAACAAA ATG AGA GAATTCC GGCC ATG CAG TGG
B / Construction of plasmids 28-56 / AC (Fig.6).

 To remove the "excess" nucleotides at the junction of the GPD promoter and the M-protein gene, the linearized restriction enzyme EcoRI was digested with GPDIV DNA of Ba131 nucleases, followed by subcloning.

10 μg of 56 / AB DNA was hydrolyzed with restriction enzyme EcoRI under standard conditions in a volume of 40 μl. After deproteinization with a phenol / chloroform mixture, DNA was precipitated with alcohol and the precipitate was dried. DNA was dissolved in 15 μl of bovine serum albumin solution (500 μg / ml) and mixed with 15 μl of 2xBa131 buffer (1.2 M NaCl, 24 mM CaCl ₂ , 24 mM MgCl ₂ , 40 mM TrisHCl, pH 8.0, 2 mM EDTA). The mixture was incubated for 10 s at 30 ^o C with 1 unit Ba131. The reactions were stopped, 0.5 μl of a 0.2 M EGTA solution was added, then treated with a phenol / chloroform mixture. DNA was precipitated with ethanol. The DNA pellet was dissolved in 5 μl of TE buffer, treated with a Klenov fragment of DNA polymerase 1 in the presence of dNTP, as described in the Maniatis manual, and doped with 100 μl of the mixture as described above. The ligase was inactivated by heating at 70 ^° C. for 10 minutes. 10 μl of 1 M NaCl and 10 units were added. EcoRI and incubated for 2 h at 37 ^o C. Plasmids from the obtained clones were analyzed by Sanger sequencing (Hattori Sakaki, 1986, Anal, Biochem, 152, 232-238) using a primer specific for the GPDI promoter sequence. The selected clone 28-56 / AC contained an intact 5'-untranslated sequence of the GPD promoter together with the ATG initiator codon and the first two codons of the glyceraldehyde phosphate dehydrogenase gene, docked in a frame, starting from the third amino acid, with the coding sequence of the M-protein gene (Fig.6) .

B / Construction of plasmids 3-28-56 / AB (Fig.7)
To construct an “expression cassette” of the HBV M protein gene, the transcriptional terminator sequence of the yeast PGK gene was attached to the 3'-end of this gene contained in plasmid 28-56 / AC so that the PGK gene coding regions were not present in the final construct, and 3'-UTR of the S-protein gene.

 Plasmid YEp64 / AB (patent application No. 4025079/13 dated 2.12.85) is an expression vector of a derivative of the HBV M protein, 20 μg of YEp64 / AB DNA were hydrolyzed together with restriction enzymes XbaI and BamHI from low-melting agarose, 870 bp were isolated. a DNA fragment containing 590 bp 3'-terminal coding region of the M protein with an intact PGKI S. cerevisiae transcription terminator attached to the stop codon.

 Similarly, an XbaI / BamHI vector of 28-56 / AC was isolated and doped with an XbaI-BamHI fragment under standard conditions. The E. coli TGI transformants were selected for the presence of the desired clone by analyzing the isolated plasmid DNA using restriction enzyme digestion; one of the obtained plasmids with the desired structure was designated 3-28-56 / AB and selected for further work.

 G / Construction of plasmids YEp63 / AB (Fig.8).

 To obtain a vector that ensures the biosynthesis of HBV M protein in yeast cells, the expression cassette from plasmid 3-28-56 / AB was cloned into the universal shuttle yeast vector 38 / AB (B. Aryaa, Cand. Diss. Moscow, 1991), which in turn, it is a plasmid pJDB207 with the yeast TRPI gene integrated into the BamHI site (in the form of a BamHI-BgIII fragment of plasmid YRp7).

10 μg of 38 / AB DNA was dihydrolyzed with BamHI restrictase enzyme under standard conditions. DNA was reprecipitated with alcohol, dissolved in 30 μl of a solution of 50 mM Tris-HCl pH 8.5, incubated with 1 unit. bacterial alkaline phosphatase (Amersham) for 30 min at 50 ^o C. A linear DNA with dephosphorylated 5'-protruding BamHI-ends was isolated from low-melting agarose.

 10 μg of plasmid 3-28-56 / AB DNA was hydrolyzed with HindII restriction enzyme to blunted using a Klenov fragment of DNA polymerase 1 to the ends of the BamHI linker DNA under standard conditions and after hydrolysis with BamHI restriction enzyme the "expression cassette" of the HBV M protein was isolated as BamHI, a DNA fragment of about 2000 bp

The dephosphorylated 38 / AB vector and the fragment were crosslinked using phage T4 DNA; transformants of E. coli TG1 strain with phenotype A were selected $\genfrac{}{}{0ex}{}{\text{R}}{\text{p}}$ T $\genfrac{}{}{0ex}{}{\text{S}}{\text{c}}$ . The presence of the insert and its orientation were determined by analyzing from the obtained plasmid clones by restriction enzyme hydrolysis. As a result, the YEp63 / AB plasmid was selected in which the 3 ′ end of the “expression cassette” was attached to the 5 ′ end of the TRP1 gene of plasmid 38 / AB, and the PGK terminator was located between the marker gene and the expressed M protein gene.

 Example 3

 Obtaining a strain of S. cerevisiae 20B12 producer of a derivative of the M-protein of HBV.

Cells of the recipient strain S. cerevisiae 20B12 (α trpl pep4-3) were transformed with YEp63 / AB plasmid DNA according to the method of Ito et al. (Ito et al. 1983, J. Bacteriol, 153, 163-168). A yeast colony from the agar plate was transferred to 50 ml of liquid YEPD medium and incubated overnight at 30 ^° C. The culture was centrifuged and the precipitate was washed with a buffer of the following composition: 10 mM Tris-HCl pH 7.5, 0.1 mM EDTA (TE -buffer). The washed cells were resuspended in 50 ml of TE-buffer containing 0.1 M LiAC and incubated for 2 hours at 30 ^° C. After centrifugation, the cells were suspended in 0.5 ml of LiOAc / TE-buffer, 10-20 μg of YEp63 / plasmid DNA was added. AB, and the mixture was incubated for 30 min at 30 ^° C. Then 1 ml of 50% PEG 4000 was added, stirred, incubated at 30 ^° C for 1 h and subjected to heat shock for 5 min at 42 ^° C. The cells were centrifuged and washed with TE buffer. The washed cells were resuspended in 500 μl of water and 100 μl were plated on Petri dishes with selective agar (0.67% YNB, 2% glucose, 2% agar). After 2-4 days of incubation at 30 ^° C, growth of transformants was observed.

To analyze the expression of the M-protein derivative in transformants of strain 20B12, clones 20B12 / YEp63 / AB were simultaneously grown and, as a negative control, strain 20B12 containing shuttle vector 38 / AB in 4 ml of minimal YNB medium containing 1% casein hydrolyzate (Difco). After culturing for 72 hours at 30 ^° C., a cell pellet was obtained from the grown cultures. A protein extract from water-washed yeast cells was obtained by shaking with periodic cooling on ice of resuspended precipitates with glass beads (0.5 mm in diameter) in 3 ml of buffer containing 50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 0.5% Triton X-100, 1 mM PMSF (10 times for 30 s at maximum speed of a table shaker type "Vortex").

To remove residual cells and beads, the homogenates were centrifuged and the suspension was clarified by repeated centrifugation. The concentration of total protein in the lysates was determined according to the Bradford method (Bradford MM 1976, Anal. Biochem. 72, 248-254). Determination of protein content with the properties of HBsAg in lysates was carried out by enzyme immunoassay. The level of expression of the HBV M-protein derivative thus determined in the 20B12 / YEp63 / AB transformants was 300 μg / L of culture medium at a culture density of 0.5 • 10 ⁷ cells / ml. One of the selected clones was deposited as a producer strain of the HBV M-protein derivative in the All-Union Collection of Microorganisms at the IBPM Academy of Sciences of the USSR under the number VKM CR-347D.

Claims (1)

 The yeast strain Saccharomyces cerevisial VKM NCR-347D containing the recombinant plasmid YEP 63 / AB producer of a derivative of the hepatitis B virus human protein M.

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