RU2172346C2 - Method of synthesis of polypeptide eliciting antigenic activity of hepatitis e virus and set for assay of antibody raised to hepatitis e pathogen - Google Patents

Abstract

FIELD: medicine, molecular biology, genetic engineering of proteins. SUBSTANCE: invention proposes a method for preparing recombinant polypeptides for their using in set for assay of antibodies raised to hepatitis E virus. Set has series of recombinant antigens synthesized in E. coli cells transformed with recombinant DNAs. Recombinant DNAs involve fragment of the second and the third open reading frames of hepatitis E virus genome. Sets can contain E. coli β-galactosidase as a control antigen. EFFECT: enhanced sensitivity and specificity in assay of spectrum of antibodies to individual viral proteins. 6 cl, 2 tbl, 13 ex

Description

 The invention relates to biotechnology and immunology, and is a method of producing recombinant proteins by synthesis in bacterial cells with antigenic activity to hepatitis E virus, and a set of proteins obtained by this method and allowing serodiagnosis of hepatitis E. Hepatitis E occurs as a result of infection of a person with hepatitis V E. The usual way to get hepatitis E virus is by drinking feces contaminated with feces; a transplacental way of infecting a child from pain is also possible. th mother. Hepatitis E is an endemic disease in countries with a hot climate, during epidemics this infection is characterized by a significant spread rate and high mortality among pregnant women (up to 25%) and newborn babies born to sick mothers (up to 77%). Therefore, to conduct emergency antiepidemic, preventive measures and timely start of treatment for hepatitis E, the development of diagnostic test systems is extremely urgent, allowing the detection of infected people in the early stages of the development of the epidemic process.

 Numerous studies of the molecular biological basis of the immune response to hepatitis E virus have made it possible to determine the structure of the viral genome and to localize the main viral antigenic determinants. Thanks to the determination of the nucleotide sequences of the genomes of viral isolates of various geographical origin and the success in studying the antigenic structure of viral proteins, the existence of two main serovariants of the virus has been proved: the Burma serotype, which includes Asian viral isolates, and the Mexico serotype, which combines American isolates.

 The initial diagnosis of hepatitis E was based on the serological exclusion of viral hepatitis of a different etiology. Most hepatitis E diagnostic methods developed to date are based on the determination of antibodies to protein products of the second and third open reading frames of the hepatitis E virus genome in blood serum (serodiagnosis). For these purposes, various approaches are used in which the principle of enzyme-linked immunosorbent assay (ELISA), immunofluorescence method, immunoblotting are used. The use of this group of methods is based on the use of synthetic peptides fragments of viral proteins or recombinant polypeptides containing fragments of viral antigens. One of the most promising ways to improve diagnostic test systems is the use of recombinant hepatitis E virus proteins synthesized in various expression systems as an antibody-binding substrate. Such recombinant proteins preserving the antigenic determinants of the hepatitis E virus are used in the serodiagnosis of the disease, providing high sensitivity and specificity of the tests used (US patent 5563032, CL 435/5, 08/10/96).

 The essence of this technical solution lies in the fact that the proposed method for producing recombinant antigens and a kit for determining antibodies to hepatitis E virus, containing as an antigen a polypeptide / polypeptides obtained by the proposed method. Recombinant antigens for hepatitis E virus of the Burmese serovariant were obtained using genetic engineering methods from E. coli producer strains. transformed with recombinant DNA containing fragments of the second and third open reading frames of the viral genome. As a control antigen, the kit may contain beta-galactosidase E. coli. In this case, the antigens included in the kit are immobilized on a solid carrier in a mixture or separately, depending on the problem being solved. Polystyrene or polyvinyl chloride beads and tablets for immunological reactions, nitrocellulose, nylon, latexes, erythrocytes, and other solid-phase carriers can serve as solid carriers. The sensitivity and specificity of the kit can be increased by introducing into the kit an additional (compared to the prototype) recombinant antigen containing an ORF2 gene product fragment from 403 to 461 amino acid residues, as well as separate sorption of various recombinant antigens, which helps to determine the spectrum of antibodies to individual viral proteins.

 The invention is illustrated by examples.

 Example 1

 Obtaining a DNA fragment of HE40.

 To obtain the DNA fragment were used oligonucleotides d1, d2, r1, r2, given at the end of the description.

The oligonucleotides are mixed and knocked out using a T4 phage polynucleotide kinase. For this, 50 pmoles of each of the oligonucleotides, 50 μl of ten-fold kinase buffer (0.5 M Tris-HCl, pH 7.6, 0.1 M MgCl, 50 mM dithiothreitol, 1 mM) are added to an 1.5 ml Eppendorf tube spermidine and 1 mM EDTA), 250 pmoles (150 μCI) [gamma-P] ATP (specific activity 3000 Ci / mol), 150 units of phage T4 polynucleotide kinase activity and bidistilled water to a volume of 500 μl and incubated for 30-60 minutes at 37 ^o C. Then the reaction mixture is heated to a temperature of 98 ^o C, cooled to 16 ^o C and mixed with DNA ligase. For this, 100 μl of a mixture of kinase-treated oligonucleotides is mixed with 12 μl of ten-fold ligation buffer (0.66 M Tris-HCl, pH 7.6, 50 mM MgCl, 50 mM dithiothreitol and 10 mM ATP), 8 μl (50 activity units) Phage T4 DNA ligases and incubated at 4 ^o C for 5 hours. Then the reaction mixture is heated to a temperature of 65 ^o C for 15 minutes to inactivate the enzyme.

 10 μl of the solution treated with kinase and ligase oligonucleotides is used for DNA analysis by electrophoresis in 12% polyacrylamide gel according to the usual method (Molecular cloning, New York, CSHL, 1982). After radio gel autography, a series of fragments larger than or equal to 92 bp is visible.

100 μl of a solution of the obtained DNA fragment is hydrolyzed with restriction enzymes BamHI and PstI in a restriction buffer solution (final concentration of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl, 1 mM dithiothreitol) for 5 hours at 37 ^o C. Enzymes are inactivated by heating at 70 ^o C.

 10 μl of a solution of DNA fragments hydrolyzed by restriction endonucleoses is used for DNA analysis in a 12% polyacrylamide gel (a fragment of about 92 bp is visible after radio-autography).

DNA is precipitated with ethyl alcohol and dissolved in 20 μl of distilled water. 10 μl of the restriction enzyme-treated DNA fragment was added to an Eppendorf tube containing 7 μl (0.1 μg / ml) of DNA vector plasmid pUC18 treated with BamHI and PstI restriction enzymes, 2 μl of ten-fold ligation buffer, 1 μl (5 units of activity) of DNA ligase phage T4 and incubated at 4 ^o C for 5 hours.

 The resulting ligase mixture transform cells of E. coli strain JM109 by the usual method (Molecular cloning. New York. CSHL, 1982). Transformed cells are plated on plates with 1.2% LB agar containing 10 μg / ml ampicillin. The grown colonies are checked for the presence of recombinant plasmids. For this, bacterial cells lyse and secrete plasmid DNA as described (Nature, 1981, 145, 1365). The isolated plasmid DNA was finalized with BamHI and PstI restriction enzymes and the hydrolyzate was examined by electrophoresis on a 12% polyacrylamide gel. A strain containing a plasmid is selected, the hydrolyzate of which has 1 DNA strip of 1686 and 92 bp on the electrophoregram, which corresponds to the size of the vector and the DNA fragment synthesized by us.

 Using the Sanger method (PNAS, 1977, 74, 5463), the nucleotide sequence of the cloned DNA fragment is determined (see the end of the description).

 Example 2

 Obtaining plasmids pHE40.

 The HE40 DNA fragment is ligated with the DNA of the vector plasmid pEL5A treated with restriction enzymes BamHI and PstI.

 The ligase mixture transform cells of E. coli strain PLT90. Transformed cells are plated on plates with 1% LB agar. containing 50 μg / ml ampicillin. The grown colonies are checked for the presence of recombinant plasmids. For this, bacterial cells lyse and secrete plasmid DNA. The isolated plasmid DNA is treated with restriction enzymes BamHI and PstI and the hydrolyzate is examined by electrophoresis in 2% agarose. Select strain SHE40. The plasmid DNA hydrolyzate from the recombinant SHE40 strain has 2 DNA bands of 6400 and 92 nucleotides on the electrophoregram, which corresponds to the dimensions of the vector and synthesized DNA fragment 40.

 When determining the nucleotide sequence of the plasmid pHE40, it was found that the synthesized fragment of the hepatitis E virus ORF2 gene through the BamHI site is attached to the 3'-end of the E. coli beta-galactosidase gene. Thus, the plasmid pHE40 encodes a recombinant protein containing the amino acid sequences of the hepatitis E virus ORF2 gene product fused to the E. coli β-galactosidase sequence.

 Example 3

 Synthesis of the HE40 polypeptide.

The E. coli strain containing the plasmid pHE40 is grown in 100 ml of LB medium at 30 ^° C. to a density of 8x10 cells / ml. After that, the temperature was raised to 40 ^o C and cells were grown for another 2 hours. Cells are harvested by centrifugation at 4000 rpm for 15 minutes. Cells are lysed by ultrasound and proteins are isolated according to the method described (EMBO J., 1984, 3, 1429).

The isolated proteins are analyzed in 10% PAG according to the standard Laemmli method (Nature, 227, 680-685, 1970). As markers of molecular masses of proteins, a Pharmacia kit containing molecular weight markers from 20 to 160 kD is used. As a control, a similarly obtained cell lysate of E. coli strain PLT90 containing vector plasmid pEL5A and without plasmids pHE40 is used. A comparative analysis of the protein composition shows that the E. coli strain containing the plasmid pHE40 expresses a polypeptide with a molecular weight of 122 kD. Below is the amino acid sequence of the polypeptide, determined on the basis of the nucleotide sequence of the cloned DNA fragment, corresponding to the product fragment of the second open reading frame from 633 to 659 amino acid residues:
X - LGLQGCAFQSTVAELQRLKMK VGKTRELQPSLLID,
where X is the amino acid sequence of beta-galactosidase E. coli.

 Example 4

 Obtaining a DNA fragment of HE60.

 To obtain the DNA fragment were used oligonucleotides d1, d2, r1, r2, given at the end of the description.

The oligonucleotides are mixed and knocked out using a T4 phage polynucleotide kinase. For this, 50 pmoles of each of the oligonucleotides, 50 μl of ten-fold kinase buffer (0.5 M Tris-HCl, pH 7.6, 0.1 M MgCl, 50 mM dithiothreitol, 1 mM) are added to an 1.5 ml Eppendorf tube spermidine and 1 mM EDTA), 250 pmoles (150 μCI) [gamma-P] ATP (specific activity 3000 Ci / mol), 150 units of phage T4 polynucleotide kinase activity and bidistilled water to a volume of 500 μl and incubated for 30-60 minutes at 37 ^o C. Then the reaction mixture is heated to a temperature of 98 ^o C, cooled to 16 ^o C and mixed with DNA ligase. For this, 100 μl of a mixture of kinase-treated oligonucleotides is mixed with 12 μl of ten-fold ligation buffer (0.66 M Tris-HCl, pH 7.6, 50 mM MgCl, 50 mM dithiothreitol and 10 mM ATP), 8 μl (50 activity units) Phage T4 DNA ligases and incubated at 4 ^o C for 5 hours. Then the reaction mixture is heated to a temperature of 65 ^o C for 15 minutes to inactivate the enzyme.

 10 μl of the solution treated with kinase and ligase oligonucleotides is used for DNA analysis by electrophoresis in 12% polyacrylamide gel according to the usual method (Molecular cloning, New York, CSHL. 1982). After radio gel autography, a series of fragments larger than or equal to 92 bp is visible.

100 μl of the solution of the obtained DNA fragment is hydrolyzed with restriction enzymes BaniHI and PstI in restriction buffer solution (final concentration of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl, 1 mM dithiothreitol) for 5 hours at 37 ^o C. Enzymes are inactivated by heating at 70 ^o C.

 10 μl of a solution of DNA fragments hydrolyzed by restriction endonucleases is used for DNA analysis in a 12% polyacrylamide gel (a fragment of about 92 bp is visible after radio-autography).

DNA is precipitated with ethyl alcohol and dissolved in 20 μl of distilled water. 10 μl of the restriction enzyme-treated DNA fragment was added to an Eppendorf tube containing 7 μl (0.1 μg / ml) of DNA vector plasmid pUC18 treated with BamHI and PstI restriction enzymes, 2 μl of ten-fold ligation buffer, 1 μl (5 units of activity) of DNA ligase phage T4 and incubated at 4 ^o C for 5 hours.

 The resulting ligase mixture transform cells of E. coli strain JM109 by the usual method (Molecular cloning. New York. CSHL, 1982). Transformed cells are plated on plates with 1.2% LB agar containing 10 μg / ml ampicillin. The grown colonies are checked for the presence of recombinant plasmids. For this, bacterial cells lyse and secrete plasmid DNA as described (Nature, 1981, 145, 1365). The isolated plasmid DNA is treated with BamHI and PstI restriction enzymes and the hydrolyzate is examined by electrophoresis on a 12% polyacrylamide gel. A strain containing a plasmid is selected, the hydrolyzate of which has 1 DNA strip on the electrophoregram 1 with sizes of 1686 and 107 bp, which corresponds to the size of the vector and the DNA fragment synthesized by us.

 The Sanger method (PNAS, 1977. 74, 5463) determines the nucleotide sequence of the cloned DNA fragment (see the end of the description).

 Example 5

 Obtaining plasmids pHE60.

 The HE60 DNA fragment is ligated with the DNA of the vector plasmid pEL5A treated with restriction enzymes BamHI and PstI.

 The ligase mixture transform cells of E. coli strain PLT90. Transformed cells are plated on plates with 1% LB agar containing 50 μg / ml ampicillin. The grown colonies are checked for the presence of recombinant plasmids. For this, bacterial cells lyse and secrete plasmid DNA. The isolated plasmid DNA is treated with restriction enzymes BamHI and PstI and the hydrolyzate is examined by electrophoresis in 2% agarose. Select strain SHE60. The plasmid DNA hydrolyzate from the recombinant strain SHE60 has 2 DNA bands on the electrophoregram of 6400 and 107 nucleotides in size, which corresponds to the sizes of the vector and the synthesized HE60 DNA fragment.

 When determining the nucleotide sequence of the plasmid pHE60, it was found that the synthesized fragment of the hepatitis E virus ORF3 gene through the BamHI site is attached to the 3'-end of the E. coli beta-galactosidase gene. Thus, the plasmid pHE60 encodes a recombinant protein containing the amino acid sequences of the hepatitis E virus ORF3 gene product fused to the E. coli β-galactosidase sequence.

 Example 6

 Synthesis of the HE60 polypeptide.

The E. coli strain containing the plasmid pHE60 was grown in 100 ml of LB medium at 30 ^° C. to a density of 8x10 cells / ml. After that, the temperature was raised to 40 ^o C and cells were grown for another 2 hours. Cells are harvested by centrifugation at 4000 rpm for 15 minutes. Cells are lysed by ultrasound and proteins are isolated according to the method described (EMBO J., 1984, 3, 1429).

The isolated proteins are analyzed in 10% PAG according to the standard Laemmli method (Nature, 227, 680-685, 1970). As markers of molecular masses of proteins, a Pharmacia kit containing molecular weight markers from 20 to 160 kD is used. As a control, a similarly obtained cell lysate of E. coli strain PLT90 containing the vector plasmid pEL5A and without the plasmids pHE60 is used. A comparative analysis of the protein composition shows that the E. coli strain containing the plasmid pHE60 expresses a polypeptide with a molecular weight of 122 kD. Below is the amino acid sequence of the polypeptide, determined based on the nucleotide sequence of the cloned DNA fragment, corresponding to the fragment of the product of the third open reading frame from 92 to 123 amino acid residues:
X - SNPPDHSAPLGVTRPSAPPLP HVVDLPQLGPRRCSQAC,
where X is the amino acid sequence of beta-galactosidase E. coli.

 Example 7

 Obtaining DNA fragment HE70.

 To obtain the DNA fragment were used oligonucleotides d1, d2, d3, d4, r1, r2, r3, r4, given at the end of the description.

The oligonucleotides d1, d2, r1, r2 are mixed and knocked out using phage T4 polynucleotide kinase. For this, 50 pmoles of each of the oligonucleotides, 50 μl of ten-fold kinase buffer (0.5 M Tris-HCl, pH 7.6, 0.1 M MgCl, 50 mM dithiothreitol, 1 mM) are added to an 1.5 ml Eppendorf tube spermidine and 1 mM EDTA), 250 pmoles (150 μCI) [gamma-P] ATP (specific activity 3000 Ci / mol), 150 units of phage T4 polynucleotide kinase activity and bidistilled water to a volume of 500 μl and incubated for 30-60 minutes at 37 ^o C. Then the reaction mixture is heated to a temperature of 98 ^o C. cooled to 16 ^o C and mixed with DNA ligase. For this, 100 μl of a mixture of kinase-treated oligonucleotides is mixed with 12 μl of ten-fold ligation buffer (0.66 M Tris-HCl, pH 7.6, 50 mM MgCl, 50 mM dithiothreitol and 10 mM ATP), 8 μl (50 activity units) Phage T4 DNA ligases and incubated at 4 ^o C for 5 hours. Then the reaction mixture is heated to a temperature of 65 ^o C for 15 minutes to inactivate the enzyme.

Oligonucleotides d3, d4, r3, r4 are mixed and knocked out using the T4 phage polynucleotide kinase. For this, 50 pmoles of each of the oligonucleotides, 50 μl of ten-fold kinase buffer (0.5 M Tris-HCl, pH 7.6, 0.1 M MgCl, 50 mM dithiothreitol, 1 mM) are added to an 1.5 ml Eppendorf tube spermidine and 1 mM EDTA), 150 units of phage T4 polynucleotide kinase activity and bidistilled water to a volume of 500 μl and incubated for 30-60 minutes at a temperature of 37 ^o C. Then the reaction mixture is heated to a temperature of 98 ^o C, cooled to 16 ^o C and mixed with DNA ligase. For this, 100 μl of a mixture of kinase-treated oligonucleotides is mixed with 12 μl of ten-fold ligation buffer (0.66 M Tris-HCl, pH 7.6, 50 mM MgCl, 50 mM dithiothreitol and 10 mM ATP), 8 μl (50 activity units) Phage T4 DNA ligases and incubated at 4 ^o C for 5 hours. Then the reaction mixture is heated to a temperature of 65 ^o C for 15 minutes to inactivate the enzyme.

The resulting DNA fragments are mixed and again subjected to treatment with T4 phage polynucleotide kinase. For this, 50 pmoles of each of the DNA fragments, 50 μl of ten-fold kinase buffer (0.5 M Tris-HCl, pH 7.6, 0.1 M MgCl, 50 mM dithiothreitol, 1) are added to the 1.5 ml Eppendorf tube mM spermidine and 1 mM EDTA), 150 units of phage T4 polynucleotide kinase activity and bidistilled water to a volume of 500 μl and incubated for 30-60 minutes at a temperature of 37 ^o C. Then the reaction mixture is heated to a temperature of 98 ^o C, cooled to 16 ^o C and mixed with DNA ligase. For this, 100 μl of a mixture of kinase-treated oligonucleotides is mixed with 12 μl of ten-fold ligation buffer (0.66 M Tris-HCl, pH 7.6, 50 mM MgCl, 50 mM dithiothreitol and 10 mM ATP), 8 μl (50 activity units) Phage T4 DNA ligases and incubated at 4 ^o C for 5 hours. Then the reaction mixture is heated to a temperature of 65 ^o C for 15 minutes to inactivate the enzyme.

 10 μl of the solution treated with kinase and ligase oligonucleotides is used for DNA analysis by electrophoresis in 12% polyacrylamide gel according to the usual method (Molecular cloning, New York, CSHL, 1982). After radio gel autography, a series of fragments larger than or equal to 182 bp is visible.

100 μl of a solution of the obtained DNA fragment is hydrolyzed with restriction enzymes BamHI and PstI in a restriction buffer solution (final concentration of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10mM MgCl, 1 mM dithiothreitol) for 5 hours at 37 ^° C The enzymes are inactivated by heating at 70 ^o C.

 10 μl of a solution of DNA fragments hydrolyzed by restriction endonucleases is used for DNA analysis in a 12% polyacrylamide gel (a fragment of about 92 bp is visible after radio-autography).

DNA is precipitated with ethyl alcohol and dissolved in 20 μl of distilled water. 10 μl of the restriction enzyme-treated DNA fragment was added to an Eppendorf tube containing 7 μl (0.1 μg / ml) of DNA vector plasmid pUC18 treated with BamHI and PstI restriction enzymes, 2 μl of ten-fold ligation buffer, 1 μl (5 units of activity) of DNA ligase . Oligonucleotides d3, d4, r3, r4 are mixed and knocked out using the T4 phage polynucleotide kinase. For this, 50 pmoles of each of the oligonucleotides, 50 μl of ten-fold kinase buffer (0.5 M Tris-HCl, pH 7.6, 0.1 M MgCl, 50 mM dithiothreitol, 1 mM) are added to the 1.5 ml Eppendorf tube spermidine and 1 mM EDTA), 250 pmoles (150 μCI) [gamma-P] ATP (specific activity 3000 Ci / mol), 150 units of phage T4 polynucleotide kinase activity and bidistilled water to a volume of 500 μl and incubated for 30-60 minutes at 37 ^o C. Then the reaction mixture is heated to a temperature of 98 ^o C, cooled to 16 ^o C and mixed with DNA ligase. For this, 100 μl of a mixture of kinase-treated oligonucleotides is mixed with 12 μl of ten-fold ligation buffer (0.66 M Tris-HCl, pH 7.6, 50 mM MgCl, 50 mM dithiothreitol and 10 mM ATP), 8 μl (50 activity units) Phage T4 DNA ligases and incubated at 4 ^o C for 5 hours. Then the reaction mixture is heated to a temperature of 65 ^o C for 15 minutes to inactivate the enzyme. The obtained ligase mixture transform cells of E. coli strain JM109 by the usual method (Molecular cloning, New York. CSHL, 1982). Transformed cells are plated on plates with 1.2% LB agar containing 10 μg / ml ampicillin. The grown colonies are checked for the presence of recombinant plasmids. For this, bacterial cells lyse and secrete plasmid DNA as described (Nature, 1981, 145, 1365). The isolated plasmid DNA is treated with BamHI and PstI restriction enzymes and the hydrolyzate is examined by electrophoresis on a 12% polyacrylamide gel. A strain containing a plasmid is selected, the hydrolyzate of which has two DNA bands 1686 and 182 bp in size on the electrophoregram. , which corresponds to the size of the vector and the DNA fragment synthesized by us.

 Using the Sanger method (PNAS, 1977, 74, 5463), the nucleotide sequence of the cloned DNA fragment is determined (see the end of the description).

 Example 8

 Obtaining plasmids pHE70.

 The DNA fragment HE70 is ligated with the DNA of the vector plasmid pEL5A treated with restriction enzymes BamHI and PstI.

 The ligase mixture transform cells of E. coli strain PLT90. Transformed cells are plated on plates with 1% LB agar containing 50 μg / ml ampicillin. The grown colonies are checked for the presence of recombinant plasmids. For this, bacterial cells lyse and secrete plasmid DNA. The isolated plasmid DNA is treated with restriction enzymes BamHI and PstI and the hydrolyzate is examined by electrophoresis in 2% agarose. Select strain SHE70. The plasmid DNA hydrolyzate from the recombinant strain SHE70 has 2 DNA bands of 6400 and 182 nucleotides on the electrophoregram, which corresponds to the size of the vector and the synthesized HE70 DNA fragment.

 When determining the nucleotide sequence of the plasmid pHE70, it was found that the synthesized fragment of the hepatitis E virus ORF2 gene via the BamHI site is attached to the 3'-end of the E. coli β-galactosidase gene. Thus, the plasmid pHE70 encodes a recombinant protein containing the amino acid sequences of the hepatitis E virus ORF2 gene product fused to the E. coli β-galactosidase sequence.

 Example 9

 Synthesis of the HE70 polypeptide.

Strain E. coli. containing the plasmid pHE70, grown in 100 ml of LB medium at 30 ^o C to a density of 8x10 cells / ml After that, the temperature was raised to 40 ^o C and cells were grown for another 2 hours. Cells are harvested by centrifugation at 4000 rpm for 15 minutes. Cells are lysed by ultrasound and proteins are isolated according to the method described (EMBO J. 1984, 3, 1429).

The isolated proteins are analyzed in 10% PAG according to the standard Laemmli method (Nature, 227, 680-685, 1970). As markers of molecular masses of proteins, a Pharmacia kit containing molecular weight markers from 20 to 160 kD is used. As a control, a similarly obtained cell lysate of E. coli strain PLT90 containing the vector plasmid pEL5A and not containing plasmids pHE70 is used. A comparative analysis of the protein composition shows that the E. coli strain containing the plasmid pHE70 expresses a polypeptide with a molecular weight of 124 kD. Below is the amino acid sequence of the polypeptide, determined on the basis of the nucleotide sequence of the cloned DNA fragment, corresponding to the product fragment of the second open reading frame from 403 to 461 amino acid residues:
X - ANGEPTVKLYTSVENAQQDKG IAIPHDIDLGESRVVIQDYDN QHEQDRPTPSPAPSRP,
where X is the amino acid sequence of beta-galactosidase E. coli.

 Example 10

 Control of antigenic activity of recombinant HE40, HE60, HE70 polypeptides.

Control of the antigenic activity of the HE40, HE60, HE70 polypeptides is carried out by immunoblotting. For this, the electrophoresis procedure is carried out (as described in Example 3), then the electrophoretically separated proteins are transferred to the BH85 nitrocellulose membranes using a 24 V, 400 mA electro-blotting apparatus for 1 hour. The quality of the transfer is checked by staining the membranes with a solution of 0.2% Ponso S in 3% TCA for 5 min at room temperature. The paint is washed twice for 30 minutes with TBS buffer (0.15 M NaCl, 20 mM Tris-HCl, pH 7.4) with 0.05% Tween-20 at room temperature. In the next step, nitrocellulose membranes with immobilized proteins are blocked with a solution of 5% skimmed milk powder in 0.1 M Na-phosphate buffer, pH 7.4, for 30 minutes at room temperature. After blocking the membrane for 1 h at 37 ^o C treated with sera containing antibodies to hepatitis E virus, at a dilution of 1: 100. Then, TBS membranes were washed three times with 0.05% Tween-20 for 30 min at room temperature. Identification of specific antibodies is carried out by incubating the filter with an antispecies peroxidase conjugate at 37 ^° C for 1 hour. Next, repeat the procedure three times washing. The reaction is checked by adding 100 μl of 30% hydrogen peroxide and 50 ml of a solution of diamnobenzidine in 50 mM Tris-HCl, pH 7.5. Analysis shows that the resulting polypeptides bind to antibodies to hepatitis E.

 Example 11

 Kit for mass serological testing of serum or plasma samples for the presence of antibodies to recombinant antigens containing protein fragments - products of the second and third open reading frames of hepatitis E.

Strains of bacteria E.coli - producers of polypeptides:
HE70 - product of the second open reading frame with amino acid sequence
ANGEPTVKLYTSVENAQQDKG IAIPHDIDLGESRVVIQDYDN QHEQDRPTPSPAPSRP
HE40 - product of the second open reading frame with amino acid sequence
LGLQGCAFQSTVAELQRLKMK VGKTRELQPSLLID
HE60 - product of the third open reading frame with amino acid sequence
SNPPDHSAPLGVTRPSAPPLP HVVDLPQLGPRRCSQAC
grown on LB medium with selective additives, cells are harvested by centrifugation, destroyed by ultrasound. Inclusion bodies containing recombinant polypeptides are washed by centrifugation and solubilized. The polypeptides are purified using chromatographic methods, the purity of the obtained polypeptides is analyzed using DDS-Na electrophoresis. In the manufacture of immunosorbent for kits, a mixture of polypeptides is adsorbed - products of the second open reading frame (the concentration of each polypeptide is 2.5 μg / ml), and the third open reading frame (5 μg / ml) dissolved in 0.1 M phosphate-buffered saline pH 7 , 6. Sorption is carried out at 4 ^o C for 18 hours.

 The horseradish peroxidase conjugate with St. Aureus Protein A or with anti-species antibodies is obtained by periodical oxidation followed by chromatographic purification of the finished conjugate. The conjugate, control sera and the buffer mixture with hydroperite (BSH) for the peroxidase reaction are lyophilized in ampoules, sealed in an inert gas atmosphere. As a control positive immunoreagent, human blood serum containing antibodies to hepatitis E virus (K +) is used. As a control negative immunoreagent (K-), human blood serum is used that does not contain antibodies to hepatitis E.

 An immunosorbent is a polystyrene or polyvinyl chloride tablet for immunological reactions with an immobilized mixture of recombinant antigens. The immunosorbent is washed three times before use. For washing, use a phosphate buffer solution of pH 7.2 with 0.5% Tween-20 (FSB-T) using an automatic or manual washer, adding at least 0.2 ml of the solution to the well. At the end of the wash, moisture is removed from the tablet.

In 88 of 96 wells, 90 μl of serum dilution solution containing a 4% ultrasound lysed E.coli cell solution is added to PBS-T and 10 μl of previously diluted (1:10) test sera are added, mixing the contents of the wells by pipetting or with a shaker. In an unfilled row, for example, in the 1st row, 100 μl of K- are added to two wells, 100 μl of K + to 4 wells and 100 μl of serum dilution solution to 2 wells (conjugate control). The tablet is sealed with a film and incubated for 60 minutes at a temperature of 37 ^o C.

The contents of the wells are removed, the plate is washed 5 times and the moisture is completely removed. 100 μl of conjugate are added to all wells of the plate. The tablet is sealed with a new sheet of film and incubated for 30 minutes at a temperature of 37 ^o C.

The contents of the wells are removed, the plate is washed 5 times and the moisture is completely removed. 100 μl of 0.05% orthophenylenediamine in a BSH solution are added to each well of the plate. The tablet is placed in a dark place for 15-20 minutes at a temperature of 15-25 ^o C. The reaction is stopped by adding 50 μl of 2N sulfuric acid to all wells.

 Registration is carried out immediately on a Multiscan-type spectrophotometer, measuring the optical density (OD) at a wavelength of 492 nm. Bringing the spectrophotometer to zero is carried out by air or on a clean, dry tablet.

 Calculate the average value of OD (OPsr) to control the conjugate, which should be no more than 0.15; Opsr K- - not more than 0.2; OPSr K + - in the range from 0.8 to 1.8. The threshold value of OD is calculated by the formula: OPsr K +, multiplied by the calculated coefficient indicated in the passport set. Serum is considered positive if the OD value of the sample is above the threshold. The sensitivity of the drug is not less than 99%, specificity is not less than 98%.

 Example 12

 A kit for repeated (specifying and differentiating) serological testing of serum or plasma samples for the presence of antibodies to each of the corresponding hepatitis E virus antigens.

The bacterial strains of E. coli, producers of polypeptides of the products of the second and third open reading frames of hepatitis E virus, as well as the producer of β-galactosidase, are grown on LB medium with selective additives. Cells are harvested by centrifugation, destroyed by ultrasound. Inclusion bodies containing recombinant polypeptides are washed by centrifugation and solubilized. The polypeptides are purified by chromatographic methods, the purity of the obtained polypeptides is analyzed by DDS-Na electrophoresis. In the manufacture of the immunosorbent for the kits, solutions of the above polypeptides and KAG (control β-galactosidase antigen) are prepared, each as a separate solution in 0.1 M phosphate-buffered saline, pH 7.6, at a concentration of 5 μg / ml, respectively. Sorption is carried out at 4 ^o C for 18 hours.

 The horseradish peroxidase conjugate with St. Aureus Protein A or with anti-species antibodies is obtained by periodical oxidation followed by chromatographic purification of the finished conjugate. The conjugate, control sera and the buffer mixture with hydroperite (BSH) for the peroxidase reaction are lyophilized in ampoules, sealed in an inert gas atmosphere.

 As a control positive immunoreagent, human blood serum containing antibodies to hepatitis E virus (K1 +) is used. As a control negative immunoreagent (K-), human blood serum is used that does not contain antibodies to hepatitis E.

 The immunosorbent is polystyrene or polyvinyl chloride tablets for immunological reactions with recombinant antigens - the products of the second and third open reading frames of the hepatitis E virus genome, and the control antigen, individually immobilized. Wells of the plate in which antigens are immobilized are called workers.

 Immunosorbent before use is washed three times with a solution of FSB-T, remove moisture from the tablet. In 88 out of 96 wells, 90 μl of serum dilution solution is added, leaving as indicated in the table. 1, empty wells for K- and K +.

 Tab. 1 shows the location of the sorbed recombinant antigens and KAG in the rows of the tablet a, b, c, d, respectively.

10 μl of pre-diluted (1:10) test sera are added to each of the 4 working wells of the horizontal row, mixing the contents of the wells by pipetting or using a shaker. In unfilled rows in accordance with table. 1 add K- and K + 100 μl to each of the eight wells. The tablet is sealed with a film and incubated for 60 minutes at a temperature of 37 ^o C.

The contents of the wells are removed using a washer, then the plate is washed 5 times and the moisture is completely removed. 100 μl of conjugate are added to all working wells of the plate. The tablet is sealed with a new sheet of film and incubated for 30 minutes at a temperature of 37 ^o C.

The contents of the wells are removed, the plate is washed 5 times and the moisture is completely removed. In each working well of the tablet, 100 μl of 0.05% orthophenylenediamine in a BSH solution are added. The tablet is placed in a dark place for 15-20 minutes at a temperature of 15-25 ^o C. The reaction is stopped by adding 50 μl of 2N sulfuric acid to all wells.

 Registration is carried out immediately on a Multiscan-type spectrophotometer, measuring the optical density (OD) at a wavelength of 492 nm. Bringing the spectrophotometer to zero is carried out by air or on a clean, dry tablet.

 A 5-well average of the OD value for K- is calculated, which should be no more than 0.2. The value of OP K + in wells with a control antigen (11H) should be no more than 0.2.

 For each serum, the OD ratios of each recombinant antigen to the OD of the control antigen are calculated. The antigen signal is considered positive if the corresponding ratio exceeds the coefficient R indicated in the passport. The signal for the antigen is considered negative if the corresponding ratio is less than the coefficient N indicated in the passport. The intermediate option is considered as doubtful.

 The sensitivity of the drug is not less than 99%, specificity is not less than 98%. As a solid carrier for applying recombinant antigens, not only immunological reaction plates can be used, but also any other solid phase. For example, polystyrene beads, nitrocellulose, latex, erythrocytes, etc. In these cases, recombinant antigens are isolated by the same method as in examples 1 and 2, the same or other immunochemical reactions (for example, agglutination) are used and the results are taken into account both spectrophotometrically and and visually or using other devices.

 Example 13

Recombinant proteins HE40, HE60, HE70 and β-galactosidase are purified as described in Example 1. Sorption of antigens on a plate is carried out at 4 ^° C, as described in examples 11 and 12. Row A of the plate is sorbed only by HE40 polypeptide; row B is sorbed with a mixture of HE40, HE60 and HE70 polypeptides; series C with a mixture of HE40 and HE60 polypeptides; row D with a mixture of HE40 and HE70 polypeptides; row E with a mixture of HE60 and HE70 polypeptides.

 11 different sera of hepatitis E patients are added to wells 1 to 11 rows of the A-E plate, and negative serum to wells 12.

 The formulation of immunological reactions and the processing of the obtained data is carried out as described in example 12. The experimental results are presented in table. 2. The (+) mark marks the wells in which the test sera reacted positively with the immunosorbent, the (-) mark marks the wells with negatively reacted sera.

 As can be seen from the table. 2, only the sorption of all four antigens (row B of the tablet) leads to the identification of all positive sera (sensitivity - 100%). In other settings, the sensitivity of the test system ranges from 82 to 90%.

 Thus, this invention allows to increase the sensitivity and specificity of the kit by determining antibodies to a greater number of viral proteins and the spectrum of antibodies to individual viral proteins.

Claims (5)

 1. A method of producing a polypeptide having antigenic activity of hepatitis E virus, comprising creating a DNA region encoding a polypeptide, cloning in vector expression to obtain recombinant DNA, transforming the obtained recombinant DNA of the host strain, culturing transformants and isolating the target product, characterized in that they create the target product a piece of DNA encoding a polypeptide containing a fragment of proteins - products of the second or third open reading frame of the viral genome.  2. The method according to claim 1, characterized in that they create a plot of DNA encoding a polypeptide containing a fragment of proteins - products of the second open reading frame of the viral genome with the amino acid sequence (see graphic part of the formula).  3. The method according to claim 1, characterized in that create a plot of DNA encoding a polypeptide containing a fragment of the protein products of the second open reading frame of the viral genome with the amino acid sequence (see graphic part of the formula).  4. The method according to claim 1, characterized in that create a plot of DNA encoding a polypeptide containing a fragment of the protein products of the third open reading frame of the viral genome with the amino acid sequence (see graphic part of the formula).  5. A kit for determining antibodies to the causative agent of hepatitis E hepatitis E virus, including an immunosorbent based on hepatitis E virus antigens, reagents for determining the presence of antibodies, characterized in that it contains fragments of proteins, the second and / or second products, as an antigen of the hepatitis E virus or a third open reading frame of the viral genome having the amino acid sequences described in claims 2-4, respectively, immobilized on a solid carrier in a mixture and / or separately.

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