RU2603056C2 - Expression plasmid vector pet32b(+)/asfv/p30e2 for production of recombinant protein, consisting of african swine fever virus protein p30, thioredoxin and polyhistidine sections - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and veterinary virology. Disclosed is an expression plasmid vector, providing production of recombinant protein in E. coli cells, consisting of african swine fever virus (ASFV) protein p30, thioredoxin and polyhistidine sections.

EFFECT: invention can be used for making virus-specific antigens for analyzing swine serums by immunoblotting.

1 cl, 3 dwg, 1 tbl, 4 ex

Description

Technical field

The invention relates to the field of biotechnology and veterinary virology, and is an expression plasmid vector that provides for the synthesis in recombinant protein of E. coli cells consisting of a p30 fragment of the ASF virus, thioredoxin (Trx-Tag) and polyhistidine sections (6xHis), and can be used for the production of virus-specific antigens in test systems designed for the study of pig sera by immunoblotting.

State of the art

ASF is a contagious, septic disease of pigs characterized by fever, signs of toxicosis, hemorrhagic diathesis and high mortality. Domestic pigs and wild boars are susceptible to ASF, regardless of breed and age, in which the disease can occur superstroit, acute, subacute, chronically and asymptomatically. The causative agent of ASF is transmitted from sick animals and surviving virus carriers by contact and alimentary routes, as well as transplacental [1]. Since vaccines against ASF have not been developed, the total slaughter of pigs in the first threatened zone with a radius of up to 20 km from the source of infection is used to eliminate and prevent the spread of the disease, as well as strict quarantine measures in the second threatened zone with a radius of 100 km. With this in mind, laboratory diagnostics are critical to making decisions about eliminating outbreaks and controlling the spread of the disease. Modern laboratory methods for the diagnosis of ASF, based on the determination of viral antigens or DNA, do not always ensure the detection of chronically or asymptomatically infected animals. In these cases, the most informative are serological methods based on the determination of antibodies to viral proteins in the blood serum of animals.

According to the recommendations of the International Epizootic Bureau (OIE), an immunoblot method should be used to confirm the doubtful results of laboratory diagnosis of ASF by indirect enzyme-linked immunosorbent assay (ELISA) or immunofluorescence reaction. It is also used in violation of the storage conditions of blood serum samples and to eliminate false-positive results in cases when blood serum antibodies from vaccinated animals react with the antigens of the cells used in the manufacture of vaccines.

In the OIE recommended test systems for detecting ASF by immunoblotting, viral antigens come from a live virus, which involves manipulating an infectious agent. The use of recombinant proteins instead of them guarantees the biological safety of the production of diagnostic test systems and the high specificity of their antigens [2, 3].

It has been established that in the early stages after infection (3-6 days) with low-, moderate- and highly virulent ASF virus strains, antibodies to the structural protein of the p30 virus are predominantly detected in pig serum [4]. It was shown that the sensitivity and specificity of ELISA using recombinant p30 to detect antibodies to ASF in the sera of experimentally infected pigs and field sera from seropositive animals with an asymptomatic course of the disease are 96% and 99%, respectively [5].

Thus, the use of recombinant p30 protein as an antigen for detecting virus-specific antibodies in ASF by immunoblotting is the preferred solution.

A known strain of E. coli M15 cells (Qiagen) with plasmid pTT9p30-9 expressing ASF virus p30 fusion protein, the yield of which upon IPTG induction was only 1 mg with 1 dm ^{3 of} bacterial culture [6], which is insufficient to obtain recombinant p30 in preparative quantities.

Patent RU 2463343 C1 discloses an E. coli BL21 (DE3) pLysS cell strain, clone pTT9 / ASFVp30 (clone 11), containing a plasmid encoding, including a recombinant protein, comprising amino acids 90 through 190 of p30 s molecular weight 14 kDa, including its conformational epitope, as well as the cellulose-binding domain. The output of the recombinant protein after purification on a cellulosic sorbent reaches 2-4 mg with 1 DM ³ bacterial culture [7].

In the description of the invention were not presented electrophoregrams of the preparations of purified recombinant protein, which would objectively indicate the absence of impurities of E. coli proteins in it, which is fundamentally important for the method of immunoblotting. In addition, the method of immunoblotting involves electrophoretic separation of polypeptides under denaturing conditions, as a result of which conformational epitopes lose antigenic activity.

The aim of the present invention is to improve the genetic construct that encodes a fragment of the p30 protein of the ASF virus and provides coverage of the maximum number of its hydrophilic sites and antigenic epitopes, as well as obtaining the most soluble form of the recombinant protein due to the leading sequence of thioredoxin included in its structure, and sequences encoding polyhistidine areas ensuring its effective purification by metal chelate chromatography in preparative quantities x.

Disclosure of invention

The technical result from the use of the present invention lies in the possibility of learning suitable for immunoblotting highly purified modified recombinant p30 ASF virus with a yield of at least 15-20 mg with 1 DM ³ bacterial culture.

To achieve the technical result of the invention, the essential set of the following steps:

1) the design design of the TrxA-6xHis-p30e2-6xHis gene, which involves the selection of oligonucleotide primers with restriction sites to flank portions of the ASF virus CP204L gene while preserving the reading frames during cloning;

2) the choice of strain E. coli and recipient plasmids to create expressing constructs;

3) optimization of the expression and chromatographic purification conditions of the recombinant protein;

4) assessment of the specificity of the recombinant protein by immunoblotting.

The leading sequence of the thioredoxin fragment in the expression vector in E. coli ensures the conservation of the recombinant protein p30e2_TrxA_6xHis (Rec p30e2) in a soluble form in the producer cytoplasm, and the presence of two 6xHis sites allows to achieve a high degree of purification of the target product on a metal chelate sorbent.

It is offered:

- the nucleotide sequence of TrxA-6xHis-p30e2-6xHis (SEQ ID No. 1), represented by the open reading frame of the chimeric protein Rec p30e2 (SEQ ID No. 3);

- recombinant plasmid pET32b (+) ASFV / p30e2 (SEQ ID No. 2), which provides a high level of expression of Rec p30e2 in Escherichia coli cells and contains the TrxA-6xHis-p30e2-6xHis gene with the sequence SEQ ID No. 1 under the control of the promoter of gene 10 of the bacteriophage T7 and T7 terminator (Fig. 1).

- amino acid sequence of the chimeric protein Rec p30e2 (SEQ ID No. 3).

The design of the TrxA-6xHis-p30e2-6xHis hybrid gene was carried out in BioEdit 7.2.5 (http://www.mbio.ncsu.edu/bioedit), taking into account the preservation of the correct reading frames when cloning into the expression vector. The nucleotide sequences encoding the corresponding regions of the ASF virus CP204L gene were obtained by PCR with specific primers on the ASF virus DNA matrix of the Stavropol2008 strain (Stavropol 01/08) [8].

To obtain the expression vector, plasmid pET32b (+) was selected, which contained the leading sequence of the E. coli thioredoxin fragment under the control of the strong promoter of the T7 phage 10 gene, which provides a high level of expression in E. coli that stably or inducibly expresses DNA-dependent RNA- phage T7 polymerase [9].

The expressing recombinant plasmid pET32b (+) ASFV / p30e2 was obtained by subcloning the nucleotide sequences of the CP204L gene of the ASF virus with primers F-p30e2 and R-p30e2 (table 1) into the pET32b (+) vector of the EcoRV restriction sites (Fig. X and EcoRV). one).

After transformation of E. coli cells of strain KRX (Promega) with plasmid pET32b (+) ASFV / p30e2 and subsequent screening of recombinants with high expression level of Rec p30e2 fusion protein, the producer strain E. coli pET32b / ASFV / p30e2 / 1 was selected.

Expression of the target protein in the obtained producer strain is carried out by cultivation on selective media with the addition of an L-ramnose inducer, subsequent collection of cell biomass, their destruction, separation of insoluble proteins and debris, and purification of Rec p30e2 protein by metal chelate chromatography under native or denaturing conditions. The final yield of the target protein is at least 15-20 mg with 1 dm ^{3 of the} bacterial culture.

The implementation of the invention

When carrying out the invention, in addition to the methods described in the following examples, a number of generally accepted methods were used, which are described in molecular biology manuals [10].

The invention is illustrated by nucleotide and amino acid sequences:

Seq ID No. 1 The nucleotide sequence of TrxA-6xHis-p30e2-6xHis encoding the chimeric protein Rec p30e2 (1 ... 327 is the leading sequence of thioredoxin (TrxA-Tag); 349 ... 366, 916 ... 933-polyhistidine sites (6xHis); 376 ... 393 - the nucleotide sequence encoding the thrombin cleavage site (thrombin site); 460 ... 474 is the nucleotide sequence encoding the enterokinase cleavage site (enterokinase site); 490 ... 894 is the nucleotide sequence encoding the recombinant p30e2).

Seq ID No. 2 The nucleotide sequence of the recombinant plasmid pET32b (+) ASFV / p30e2 (746 ... 1072 - the leading sequence of thioredoxin (TrxA-Tag); 140 ... 157, 707 ... 724 - polyhistidine regions (6xHis); 680 ... 697 - nucleotide sequence, coding thrombin cleavage site (thrombin site); 599 ... 613 - nucleotide sequence encoding the enterokinase site (enterokinase site); 179 ... 583 - nucleotide sequence encoding the recombinant p30e2).

Seq ID No. 3 Amino acid sequence of the chimeric protein Rec p30e2 (1 ... 109 - leading sequence of thioredoxin (TrxA-Tag); 117 ... 122, 306 ... 311 - polyhistidine sites (6xHis); 126 ... 131 - thrombin cleavage site (thrombin site); 154 ... 158 is the enterokinase cleavage site (enterokinase site); 164 ... 298 is the sequence of recombinant p30e2).

The invention is illustrated by graphic materials.

FIG. 1 - Scheme of the recombinant plasmid pET32b (+) ASFV / p30e2. Notes: the position of the nucleotide insert "p30e2" is indicated; arrows indicate open reading frames of the TrxA-6xHis-p30e2-6xHis gene; sites of enterokinase and thrombin "EC and Trb"; restriction sites; "T7_promoter" - promoter of phage T7; "T7_terminator" is the sequence of the terminal portion of the T7 phage; "AmpR" - bla (β-lactamase) gene encoding resistance to ampicillin, carbenicillin and other derivative antibiotics; "PBR 322_origin" - initiation of ColE1 / pMB1 / pBR322 / pUC replication.

FIG. 2 - Electrophoregrams of the original E. coli cell lysate (producer strain KRX pET32b / ASFV / p30e2 / 1) (1), fractions during washing of the sorbent (2-4) and fractions of the purified recombinant protein (5). M - stained molecular weight markers of Spectra ™ Multicolor 10 - 260 kDa proteins.

FIG. 3 - Specific staining of the immunostrip (indicated by the arrow) as a result of the interaction of the recombinant p30 protein of the ASF virus with specific antibodies of the positive control serum of the pig immune to the ASF virus (K ⁺ ), and the absence of staining of the immunostrip after incubation with the blood serum of the intact pig (K ^- ). Note: K ⁺ is a positive inactivated control serum, K ^- is a negative control serum.

The invention is illustrated by the following examples.

Example 1. Obtaining a specific nucleotide insert that encodes a portion of the gene CP204L (p30) ASF virus

The amino acid sequence of the p30 structural protein of ASF virus is 201 amino acids (CP204L gene). At the first stage, bioinformatics analysis was carried out to search for transmembrane regions and / or hydrophobic regions that could adversely affect protein expression or subsequent purification. Using the algorithm for the search for extended hydrophobic sites “Noor and Woods” (BioEdit 2.7.5), as well as the TMNMM Server 2.0 server (http://www.cbs.dtu.dk/services/TMHMM/), 2 overlapping sites are localized cloning.

The nucleotide sequence of the ASF virus genome of Georgia / 2007 strain (GeneBank FR682468.1) was chosen as an object for primer calculation. When selecting primers, the minimum content of dimeric structures and self-complementary regions was observed, which can lead to the formation of hairpins with a high melting point. EcoRV and XhoI restriction sites are included in the primers F-p30e2 and R-p30e2. These restriction sites allow the nucleotide insertion to be cloned into the desired prokaryotic and eukaryotic expression vector. The sequence of the calculated oligonucleotide primers are shown in table 1.

PCR was performed on a Tertsik MS2 instrument (DNA Technology, Russia). The genomic DNA of the ASF virus of the Stavropol strain 01/08 (Stavropol2008) circulating in the Russian Federation was used as a matrix [8]. Preparative reactions were carried out in a volume of 25 μl. The reaction mixture consisted of: 1x buffer for thermostable pfu DNA polymerase; 10 pmol of each primer; 2.5 mM mixture of deoxyribonucleotide triphosphates; 2.5 units pfu polymerase. On top of this mixture was layered 20 μl of mineral oil.

Amplification mode of the CP204L gene region (p30): hot start: 95 ° C - 3 min × 1 cycle; 24 cycles: denaturation 95 ° С - 30 sec, annealing 54 ° С - 30 sec, elongation 72 ° С - 30 sec; final elongation: 72 ° C - 3 min × 1 cycle.

PCR products (amplifications) were isolated from 1% agarose gel using the GeneJET Plasmid Miniprep Kit (Thermo Scietific, USA) according to the manufacturer's protocol.

Purified PCR products were digested with the corresponding endonucleases for 1 hour at 37 ° C: EcoRV + XhoI in Red buffer (Thermo Scietific, USA) according to the recommended manufacturer's protocol. The restriction products were subjected to fractionation in a 1% agarose gel, followed by isolation with the GeneJET Gel Extraction Kit (Thermo Scietific, USA).

Example 2. Construction of expressing plasmid vector pET32b (+) ASFV / p30e2

The acceptor plasmid pET32b (+) (Novagen) was digested with EcoRV and XhoI endonucleases in Red buffer for 4 hours at 37 ° C. Upon completion of the restriction, the endonucleases were inactivated by heating at 80 ° C for 20 minutes. The entire volume was added to the wells of a 1% agarose gel for subsequent electrophoresis. Linearized plasmid ~ 5900 bp in size isolated from the gel using the GeneJET Gel Extraction Kit.

To obtain the plasmid pET32b (+) ASFV / p30e2, the purified restricted PCR product obtained using the primers F-p30e2 and R-p30e2 was ligated with the linearized plasmid pET32b (+) restricted by EcoRV / XhoI. Ligation was carried out using T4 DNA ligase in the recommended buffer (“α enzyme”, Russia) at room temperature (25 ± 2) ° С for an hour, the ratio of insert to vector was 2: 1.

The E. coli strain KRX cells with the genotype [F ′, traD36, ΔompP, proA ⁺ B ⁺ , lacI ^q , Δ (lacZ) M15] ΔompT, endA1, recA1, gyrA96 (Nal ^r ), thi-1, were transformed with the obtained ligase mixture. _{^{hsdR17 (r k -, m k}} +), e14 - (McrA), relA1, supE44, Δ (lac-proAB), Δ (rhaBAD): T7 RNA polymerase.

For this, 3 μl of the ligase mixture was added to 50 μl of a suspension of E. coli cells, incubated on ice for 10 minutes, subjected to thermal shock (42 ° C for 20 sec), cooled, and then the suspension was transferred to a Petri dish with a solid agarized nutrient medium containing ampicillin at a concentration of 100 μg / cm ³ , followed by incubation in an incubator at 37 ° C for 18 hours. Single colonies were analyzed by PCR using a DNA template of clones and primers used to obtain the PCR product. Clones positive for PCR were expanded in 3 cm ^{3 of} SOB nutrient broth (ampicillin 100 μg / cm ³ ) and plasmid DNA was isolated using the GeneJET Plasmid Miniprep Kit reagent kit according to the manufacturer's protocol (ThermoScientific). Sequencing for verification of structures was performed according to the Sanger method. As a result of sequencing, it was found that in the insertion region of the plasmid pET32b (+) ASFV / p30e2 of the producer strain pET32b / ASFV / p30e2 / 1, the mutation was not contained, i.e., the correct TrxA-6xHis-p30e2-6xHis gene sequence is encoded. A map of the expression plasmid construct is shown in FIG. 1, its nucleotide sequence is SEQ ID No. 2. The amino acid sequence of the expression product is shown in SEQ ID No. 3.

The resulting recombinant protein p30 of the Stavropol strain 01/08 (Stavropol2008) Rec p30e2 encodes a sequence from 56 to 190 amino acids and corresponds to that of other ASF virus isolates circulating in the Russian Federation.

Example 3. Induction of expression and chromatographic purification of protein Rec p30e2

The producer strain pET32b / ASFV / p30e2 / 1 was seeded from aliquots in 20 cm ^{3 of} liquid SOB medium containing 100 μg / cm ³ ampicillin, cultured on a thermo shaker for 14 hours at 37 ° C ("night culture") MR Green & J. Sambrook (2012) [10].

The next day, “night culture” was reseeded on a nutrient medium in a medium to seed ratio of 25: 1, respectively, and cultured according to the method of FW Studier et al. (2005) [11] in SOB-5052 medium with 50 μg / cm ^{3 of} ampicillin and using a shaker for 2-3 hours at a temperature of (37,0 ± 0,5) ° C and moderate shaking (110-120 rev / min) to a concentration of (6-7) × ¹⁰ July viable cells in cm ^3, the optical density of the bacterial culture at a wavelength of 600 nm (OD ₆₀₀ ) was 0.4–0.5 (the procedure for growing the “night” seeded onto freshly prepared medium bacterial culture). Then the bacterial culture was incubated for 1-2 hours to a concentration of (7-9) × 10 ⁷ viable cells in cm ³ (OD ₆₀₀ 0.5-0.6) at a temperature of (28.0 ± 0.5) ° C and also with constant shaking, then, the inducer of expression of the recombinant protein — 20% L-rhamnose solution — was introduced in a ratio of 1: 200 (5 cm ^{3 of the} inductor / 1000 cm ^{3 of the} bacterial culture) [9, 11, 12], and continued to incubate with the same parameters for another 16-18 hours (“auto-induction” of expression of the recombinant protein Rec p30e2).

For work, we used a chelating sorbent from a hydrophilic matrix - Ni ²⁺ -sepharose (HIS-Select Nickel Affinity Gel, Sigma), pH of buffer solutions in the chromatographic system 7.8 ± 0.2.

To prepare the cell lysate, the bacterial culture of the recombinant clone was transferred to 50 cm ³ pre-weighed polypropylene tubes, the bacterial culture was cooled for 1 hour in a domestic refrigerator at 4 ± 0.5 ° С, the bacterial culture cells were besieged by centrifugation at 3400 g for 30 minutes and a temperature of 4 ± 0.5 ° C. The supernatant was removed and the pellet was frozen. After freezing, a lysate was prepared, for which the cells were resuspended based on 1 g of a precipitate of 10 cm ^{3 of} lysis buffer solution and transferred to 4 cm ³ glass vials. Biomass was destroyed by three series of ultrasonic treatment with constant cooling of the mixture at a frequency of 18 kHz, each series - three times for 40 seconds with a break of 1 minute, with a break between series of 15 minutes. The scoring process was microscopically monitored by staining aliquots of the lysate with fuchsin Pfeifer. Cell debris was removed by centrifugation on a benchtop centrifuge at 13,000 g for 5 minutes. The supernatant was collected and mixed with a double buffer solution for application in a ratio of 1: 1, adjusting the pH of the sample to a value of 7.8 ± 0.2. The total protein content in the lysate was determined by O.N. Lowry et al. (1972) [13]. The sample was filtered through membrane filters Millipore 0,45 microns in glass vials of 10 cm ^3.

Chromatographic purification of the recombinant protein was carried out at a constant flow rate of solutions with a 0.5 cm ³ / min peristaltic pump. To elute the recombinant protein, a buffer solution (pH 7.8 ± 0.2) was used: PBS with 300 mM NaCl; 500 mM Imidazol; 0.25 mg / cm ³ Pefablock SC (Roche). The purified recombinant protein fraction was dialyzed in PBS with 300 mM NaCl and 0.25 mg / cm ³ Pefablock SC (Roche) (pH 7.8 ± 0.2) for 16-18 hours at a temperature of (4.0 ± 2, 0) ° C.

The recombinant protein solution was aliquoted and stored frozen in a domestic or low temperature refrigerator.

The purification efficiency of the recombinant protein was evaluated in 10% SDS-PAGE (SDS-PAAG) electrophoresis according to UK Laemmle (1970) [14] (Fig. 2). The yield of purified recombinant Rec p30e2 was at least 15.0-20.0 mg with 1 dm ^{3 of the} bacterial culture, which is five times higher than when using the prototype.

Example 4. The assessment of the specificity of the recombinant protein Rec p30e2 by immunoblotting

Dilutions of chromatographically purified Rec p30e2 protein were subjected to fractionation in 10% SDS-PAG (SDS-PAAG) electrophoresis according to U.K. Laemmle (1970) [14] followed by the electric transfer of polypeptides from a polyacrylamide gel to a nitrocellulose membrane in a semi-dry buffer system according to the methods of J. Kyhse-Andersen (1984) [15]. The membrane after electrotransfer was blocked with a 2.0% solution of skimmed milk powder in a phosphate buffer solution with 0.1% Tween-20 (FBI-t) (pH 7.8 ± 0.2) at 4 ± 0.5 ° C in for 16-18 hours, after which the membrane was cut into single tracks.

The immunoblotting reaction was carried out based on the methods of the OIE [2, 3].

The parameters of the immunoblotting are selected as follows:

- incubation of immunostripses for 1.5 hours at (25.0 ± 2.5) ° С with the studied sera at 1:20 dilutions or with the studied 20% suspensions of organ samples on FBI-t;

- three-time washing of test strips in the FBI-t for 5 minutes at (25.0 ± 2.5) ° С (total washing time is 15 minutes);

- incubation of immunostripses for 45 min at (25.0 ± 2.5) ° С in the working dilution of the peroxidase conjugate with protein A (working dilution of the conjugate 1: 2000);

- four-time washing of test strips in the FBI-t for 5 minutes at (25.0 ± 2.5) ° C (total washing time is 20 minutes);

- staining of immunostrips in a solution of a chromogenic substrate for 10-15 s at (25.0 ± 2.5) ° С;

- stopping the reaction by immersion of the immunostrips in distilled water;

- visual recording of reaction results.

When taking into account the results of each comparison performed immunostripa negative (K ^-) and positive (K ⁺⁾ controls. The reaction is considered positive if a colored band of varying intensity and width is detected at the level of a positive control in the center of the immunostrip, negative - in the absence of specific staining (Fig. 3).

These results showed that the presence of the thioredoxin sequence in the recombinant Rec p30e2 protein does not affect the specificity of serological reactions when using the indicated protein as an antigen, and this also simplified the procedure of isolation and chromatographic purification.

Information sources

1. Sanchez-Vizcaino, J.M. (2006). African swine fever. In Diseases of Swine, 9 Edition, pp 93-102. Ed A.D. Leman, B.E. Straw, W.L. Mengeling, S Dallaire and D.J. Taylor Iowa State Univesity Press, 2000 .-- P. 159-165.

2. Immunoblotting OIE for Serological Diagnosis of African Swine Fever (SOP / CISA / ASF / IB / 1/2008) [Electronic resource]. - Access mode: http://asf-referencelab.info/asf/images/files/SOPs/SOP-AFSIB12008.pdf. - Zagl. from the screen.

3. Manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees). Capter 2.8.1. African swine fever / Office International des Epizooties [Electronic resource]. - Paris, France, 2008 .-- 6th ed. - Access mode: http://www.oie.int/fileadmin/Home/fr/Health_standards/tahm/2.08.01_ASF.pdf. - Zagl. from the screen.

4. Sereda, A.D. Proteins of the African swine fever virus / A.D. Sereda, D.V. Kolbasov // Scientific journal KubSAU. - 2012. - No. 77 (03). - S. 21-37. - 0421200012 \ 0181. http://ej.kubagro.ru/2012/03/pdf/48.pdf.

5. Serodiagnosis of African swine fever using the recombinant protein p30 expressed in insect larvae / M.G. Barderas, A. Wigdorovit, F. Merelo, F. Beitia, C. Alonso, M.V. Borca, J.M. Escribano // Journal of Virological Methods. - 2000. - Vol. 89, N 1-2. - P. 129-136.

6. Kopytov, B.O. Cloning and expression of genes for structural proteins p30 and p72 of the virus of African swine fever: dis .... Cand. biol. Sciences: 03.00.06: protected 12.23.04 / Kopytov Valeriy Olegovich. - Pokrov, 2004 .-- 140 s.

7. Pat. RU 2463343 C1. The strain of E. coli cells BL21 (DE3) pLysS, clone pTT9 / ASFVp30 containing a recombinant plasmid with the insertion of a portion of the gene for African swine fever virus CP204L encoding the p30 protein conformational epitope for the manufacture of diagnostic preparations / B.O. Kopytov, S.Zh. Tsybanov, A.S. Kazakova, T.E. Yuzhuk, S.A. Belyanin, A.G. Guzalova, N.N. Vlasova, D.V. Sausages (RU). - No. 2011141517/10; declared 10/13/2011; publ. 10/10/2012, Bull. No. 28. - 8 p. Access mode http: www.freepatent.ru/images/patents/54/2463343/patent-2463343.pdf.

8. Pat. RU 2439152 C1. The strain "Stavropol 01/08" ASF virus for virological, molecular genetic and monitoring studies / V.M. Balyshev, D.V. Kolbasov, V.V. Kurinnov, Yu.F. Kalantaenko, S.Zh. Tsybanov, A.N. Zhukov, A.P. Vasiliev (RU). - No. 2010126641/10; declared 06/30/2010; publ. 01/10/2012, Bull. No. 1. - 6 p. Access mode: http://www.freepatent.ru/images/patents/19/2439152/patent-2439152.pdf.

9. Novagen PET System Manual. - 11th Edition. User Protocol TB055 Rev. C 0611 JN 1-63X [Electronic resource]. - Access mode: http://www.emdmillipore.com/chemdat/en_CA/Merck-US-Site/USD/ViewProductDocuments-File?ProductSKU=EMD_BIO-71867&DocumentType=USP&DocumentId=/emd/biosciences/userprotocols/en-US/Tervices .pdf & DocumentSource = GDS. - Zagl. from the screen.

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Claims (1)

The expression plasmid vector pET32b (+) ASFV / p30e2 (SEQ ID NO: 2), the structural embodiment of which is disclosed in FIG. 1, consisting of plasmid pET32b, a fragment of the gene for African pig plague virus p30 protein CP204L and two polyhistidine sequences, which provides for the synthesis of recombinant protein Rec ID p30e2 (SEQ ID No. 1) SE ID No. 1 SEX ID No. 1 SEX ID NO. 3) used as a virus-specific antigen for the diagnostic study of pig sera by immunoblotting.

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