SU1751208A1 - Recombinant plasmid dna pfr blv-f6, encoding bacteriophage @@ envelope protein and protein @@51 of cattle leukosis virus, method of its construction and strain of bacteria escherichia coli - a producer of bacteriophage @@ envelope protein and protein @@51 of cattle leukosis virus - Google Patents

Abstract

This invention relates to the microbiological industry and biotechnology. The aim of the invention is to obtain a fusion protein having the activity of the bacteriophage fr shell protein and cattle leukemia virus protein 51. The recombinant plasmid DNA pFRBLV-F6 is obtained, which consists of the vector plasmid pFR 20 carrying the protein gene of the bacteriophage fr shell under the control of the tryptophan promoter promoter , and a fragment of the gene dr 51 bovine leukemia virus, 147 bp long. the EcoRV site introduced into the envelope protein gene, the corresponding second amino acid of the envelope protein, with the translation of both the other 51 gene and the envelope protein gene, the white fr CP-BLVflp 51 consists of 130 amino acids of the envelope protein and 48 amino acids of the other 51 and has antigenic properties of both proteins. The obtained strain E coli K802, carrying the plasmid pFRBLV-F6. 3 sec. f-ly, 2 tabl

Description

This invention relates to the microbiological industry and biotechnology.

The aim of the invention is to obtain a fusion protein having the activity of the bacteriophage fr coat protein and the protein of other 51 cattle leukemia virus.

A recombinant plasmid pFRBLV-F6 was constructed, which consists of the following elements:

The DNA plasmid pRF20 harboring the complete gene of the bacteriophage frc introduced by the second amino acid at the unique restriction site EcoRV and deletion in the gene of tetracycline resistance, length 5279 bp,

Bglll-BamHI DNA fragment I HVI, encoding a portion of the sequence of others 51 (49 amino acids), corresponding to 56-103 amino acids of mature others 51 and containing a neutralizing epitope with adjacent amino acids and an immunological epitope, the 147 bp monoclonal antibody recognition site.

8 DNA composition of recombinant plasmid pFRBLV-F6 includes genes

gene fr CP-BLV dr 51 providing the synthesis of recombinant protein,

h

SL

YU

about with

gene Na providing ampicillin resistance. The CP-BLV gene fr dr 51 is under the control of the trp promoter.

Plasmid pFRBLV-F6 is amplified by addition of chloramphenicol to the medium, non-conjugative.

The essence of the method for constructing the plasmid pFRBLV-F6 is that a fragment of the other gene 51 of the leukemia virus is inserted into the second amino acid of the fr shell protein to form the recombinant frCP-BLVflp51 gene.

The producer strain is obtained by transformation of E. coli cells. Recombinant strain pFRBLV-F6 plasmid DNA.

The strain is characterized by the following features.

Morphological features: cells rod-shaped, gram-negative.

Cultural characteristics: cells grow on commonly used nutrient media, form medium-sized colonies.

Physical and biochemical characteristics: the optimum cultivation temperature is 37 ° C, the optimum pH is 7.0-7.4. Carbohydrates are used as carbon sources, mineral salts are used as nitrogen sources, as well as organic compounds in iide peptone, tryptone, amino acids,

Antibiotic resistance4 is resistant to ampicillin due to the presence of a plasmid. The presence of plasmid DNA in the strain is confirmed by testing the resistance to ampicillin, as well as by isolating and analyzing the plasmid DNA by an express method.

The productivity of the strain is 0.5% of the target product of total cellular protein.

The strain is deponated in the collection of the Central Museum of Industrial Microorganisms under the number VKPM B-4984.

Example 1. Construction of recombinant plasmid DNA pFRBLB-F6.

20 µg of plasmid containing the Bglll-BamH fragment of others 51 and isolated by the standard method, 50 units are cleaved. restrictase Bglll and 50 units. restriction enzymes BamHI in 100 μl of solution A containing 0.1 M Tris-HCl, pH 7.5, 0.05 M NaCI and 0.01 M MgCh for 2 hours at 37 ° C. Restriction enzymes inactivate 15 min by heating at 65 ° C. After purification of the DNA of the Bgltl-BamHI fragment on an agarose gel, the DNA is dissolved in 20 µl of water. The filling of the ends is carried out in 100 µl of Solution B containing 0.05 M Tris-HCl. pH 7.5, 0.01 MgCl2, 0.01 M dithio-Reitol, 0.025 M NaCI, 100 μM dATP, dCTP, dTTH and dGTP and 10 u. E. coli DNA polymerase (Klenow fragment), for 1 h at

12 ° C. After reprecipitation with ethanol, the DNA is dissolved in 10 µl of water (DNA 1).

2 µg of the plasmid pFR 20 cleaves 3 units. restrictase EcoRV in 15 µl of solution B containing 0.01 M tris-HCl, -pH 7.5, 0.05 M NaCI and 0.01 M MgCl2, for 1 h at 37 ° C, put the incubation mixture at 1 , 5% agarose gel. The fragment corresponding to the linearized plasmid 0 pFR 20 is isolated from an agarose gel (DNA 2),

0.1 μg of DNA 1 and 0.1 μg of DNA 2 are crosslinked with T4 DNA ligase in 10 μl of solution G containing 0.05 M Tris-HCl, pH 7.5, 0.01 M 5 MgCla. 0.01 M dithiothreitol, 50 μM ATP and 10 units. T4 DNA ligase, for 12 h at 4 ° C. The enzyme is inactivated by heating at 65 ° C for 15 minutes. To the reaction mixture, 100 µl of E. coli RR1 cells, 0 treated with 0.1 M CaCh (final concentration 5x10 cells / ml) were added to transform the cells.

The selection of clones is carried out by the express selection and sequencing of 5 plasmid DNA, isolated by the express method. The plasmid with the expected sequence is given the name PFRBLV-F6.

Strain producing E. coli K802 (pFRBLV-0 F6).

The producer strain is obtained by transformation of E. coli K802 cells with the recombinant plasmid pFRBLV-F6. Cells are grown in a saline medium M9 with the addition of, g / l: kazami-5nova acids 10, glucose 2, ampicillin 0.02 to the optical density of Opeco 4-5.

Identification of other 51- and fr-activity by the method of immunoblotting.

For immunoblotting, cells (6 mg) are suspended in 100 µl of Laemmli polyacrylamide gel sample buffer containing 2% dodecyl sodium sulfate (SDS) and 2% / mercapto, Chanol. , and lysed for 2 min by heating in a 5-boiling water bath. The resulting lysates (2-4 mg / ml protein) were applied to a polyacrylamide gradient gel (12-23%) with a size of 150x150x0.75 mm. Electrophoresis Conducted for 15 hours at a current of 7 mA. 0 After electrophoresis, proteins are transferred to nitrocellulose.

Filters are incubated with monoclonal anti-drug 51 gpAK 14 at a dilution of 1: 500 on TBS buffer containing 5 50 mM Tris-HCl, pH 7.5, 0.15 M NaCI, 0.1% Triton X100, 1% BSA, for 15 h at 20 ° C. After washing three times with TBS buffer, the filters are incubated for 2 ° h at 20 ° C with a peroxidase conjugate with antibodies against mouse immunoglobulins in dilution

1: 500. The filters are washed with TBS buffer (3-5 times) and develop with diaminobenzidine. In a parallel setting, the filters are incubated with rabbit anti-fr CP antibodies at a dilution of 1.200 on TBS buffer for 15 hours at 20 ° C. After washing twice with TBS buffer, the filters are incubated for 2 hours at 20 ° C with peroxidase conjugate with protein A S. aureus. The lysates of the producer strain cells in both cases reveal specific staining zones corresponding to the protein with mol.m. 19.32 kDa (or 184 amino acids long). Control lysates of E. coli K802 cells do not detect such zones.

Example 2. Determination of other51 activity in the composition of the fr CP-B1U f 51 fusion protein by the enzyme immunoassay method on the solid phase.

96-well polystyrene plates for immunological reactions are used as the solid phase. Purified fr protein CP-BLV et al 51 is diluted to a concentration of 10 µg / ml in 50 mM Tris-H.CI, pH 8.0, 0.15 M NaCI and tons per well of tablets - 100 µl each. Incubate for 1 h at 37 ° C, after which the solution is removed from the wells, the plates are washed 4-5 times with distilled water and dried. Monoclonal anti-viral antibodies (mice) or polyclonal anti-fr antibodies (rabbit) are introduced into the wells at various dilutions. After incubation for 1 h at 37 ° C, the plates are washed with distilled water and 100 µl of horseradish peroxidase konyogata with antibodies against rabbit or mouse immunoglobulins are added at a dilution of 1: 500. After incubation for 1 hour, the plates are washed and the color reaction is carried out. 100 μl of a 0.25% orthophenylene diamine solution, 2 M HCI in OJ M sodium citrate, pH 5.0, 0.02% NaOa, are incubated in the wells for 30 min at 20 ° C, the reaction is stopped by adding 100 µl 0.1 H.HCI. The appearance of a brown color indicates the presence of antigenic activity; optical density is measured at 492 nm to quantify the activity (Table 1). The absence of the reaction in the negative control (fr CP) indicates the binding specificity of anti-others 51 (antibodies recombinant protein fr CP-BLV others 51.

Example 3. Immunogenic activity of the fr protein of CP-BLV et al 51.

To determine the immunogenicity, recombinant fr protein CP-BLV et al 51 immunizes rabbits. To do this, mix 2 mg of protein, dissolved in 1 ml of physiological PBS, with 1.25 ml of Freund's complete adjuvant and get a homogeneous emulsion. Protein is injected subcutaneously. Antigen injections are repeated on days 14 and 21 and they begin to take blood from 28 days. Specificity of antibodies is determined by ELISA using two antigens. 1 fr

5 CP (for the determination of anti-fr CP antibodies), HB with Ad - other 51 (for the definition of another 51 avitel). Antibody titers on day 28 of immunization are given in Table. 2

Thus, the invention allows

0 to obtain a fusion protein of the shell of an RNA-containing bacteriophage fr and the shell of the leukemia rus in cattle, which has immunological activity both for the bacteriophage fr protein and

5 squirrel dr 51 bovine leukemia virus.

Claims (3)

Invention Formula 0 1. Recombinant plasmamdna DNA pFRBLV-F6, coding bacteriophage fr coat protein and protein 51 of cattle leukemia virus, 5426 bp in size, mol.m. 3.48 MDa containing five . EcoRV-EcoRV - DNA fragment of plasmid pFR 20, size 5279 bp, Bglll-BamHI -fragmentDHKAHV1 encoding a protein of others 51 cattle leukemia virus and a coat protein 0 RNA-containing bacteriophage fr, size 147 p., unique restriction sites with coordinates: five 0 BspMII- Pstl-Scal - Hpal-Mstll - Sacl-Aval O (origin) 1947 2182 3640 3885 4580 4801, genetic markers and regulatory sites: Na gene, providing resistance to ampicillin coordinates 1629-2489, Ptrp - promoter coordinates 3635-2648. 2. Method for constructing recombinant plasmid DNA pFRBLV-F6 encoding bacteriophage envelope protein fr and protein 51 of the bovine leukemia virus, including DNA hydrolysis with Bglll and BamHI restrictases, treatment of hydrolyzate with E. coli DNA polymerase, ligation of DNA fragments with DNA plasmid pFR 20, restriction enzyme-treated EcoRV; transformation of E. coli RRI cells with the resulting ligase mixture; selection by sequencing of clones carrying the FRBLV-F6 plasmid. 3. The bacterial strain Escherlchla coll VKPM B 4984-producing protein shell bacteriophage fr and protein dr 51 of cattle leukemia virus. Table 1 5. T and l and c and 2 Immunogenicity of recombinant protein fr CP-BLVgp 51