DD286817A5 - PROCESS FOR PREPARING THE RECOMBINANT PROTEIN FRCP-BLVGP51SH THAT CONTAINS A FUSION FROM COAT PROTEIN OF THE PHAGEN FR AND THE BOVINE LEUKAEMIEVIRUS HIP PROTEIN SECTION GP51SH - Google Patents

Abstract

The invention relates to a method for the production of the recombinant protein frCP-BLVgp51sh, which represents a fusion of coat protein of the phage fr and the envelope protein section gp51sh of the bovine leukemia virus. The essence of the invention is that the fragment of the BLV env gene, designated BLVgp51sh, which encodes amino acids 56 to 64 of the main mature wholegrain protein, into the codon for the second amino acid of phage coat protein to produce the recombinant gene frCP-BLVgp51sh is installed. The producer strain of the frCP-BLVgp51sh is obtained by transformation of E. coli K802 cells with the recombinant plasmid DNA pFRBLVsF6. {Kapsid; Coatprotein; Phage fr; Bovine Leukemia Virus; coat protein; epitope; recombinant DNA; genetic engineering; Escherichia coli; Diagnostic Virology; Vaccine}

Description

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Area of application of the invention

The invention relates to the recombinant protein frCP-BLVgpBish, which represents a fusion of coat protein of phage fr and envelope protein portion gp51sh corresponding to the sequence between 56th and 64th amino acids of mature gp51.

Characteristic of the known state of the art

Recombinant structures carrying epitopes of bovine leukemia virus (BLV) gp51 are known to be derived from bacterial proteins (1). Their disadvantages are the low immunogenital and the difficulty of purifying the recombinant proteins, which is due to the large proportion of cellular protein.

The recombinant structures on the basis of the coat protein of the RNA-containing bacteriophage fr have a number of advantages over traditional hybrid proteins based on β-galactosidase, β-lactarnase, chloramphenicol-acetyltransferase etc. Even in the absence of spontaneous self-assembly of the monomeric subunits to capsids the coat protein of phage fr, but also its analogues, by a high immunological activity and also by better cleaning options.

Object of the invention

The object of the invention is the production of a recombinant protein having immunological activities of the FR coat protein and the outer envelope protein gp51 of the BLV.

Explanation of the essence of the invention

The object of the invention is to obtain a recombinant protein having the immunological activities of fr coat protein and the epitope BLVgp51sh corresponding to the section between 56th and 64th amino acids of the mature gp51, and to provide a producer strain for this recombinant protein.

This object is achieved according to the invention by obtaining, as a result of the construction of the recombinant gene frCP-BLVgp51sh, which codes for the synthesis of the corresponding protein, and the expression plasmid pFRBLVsF6, a producer strain E. coli (pFRBLVsF6) which inhibits the production of the recombinant protein frCP- BLVgp51 sh saves. The recombinant plasmid pFRBLVsF6, whose design scheme is shown in Figure 2, consists of the following elements:

- DNA of the plasmid pFR 20 (2), which has the complete coat of the phage fr and contains a deletion in the gene for tetruclin resistance

Fragment of the BLV genome HUI (3) which corresponds to the section between the restriction sites BglII and Eco78I and encodes the region of the mature gp51 (56th to 64th amino acid) which contains the recognition site for the mAb monoclonal antibody 14.

The DNA of the recombinant plasmid pFRBLVsF6 contains the genes:

Gene frCP-BLVgp51sh, which ensures the synthesis of the recombinant protein,

- Gene bla, which confers ampicillin resistance.

Gon frCP-BLVgp51sh is under the control of a promoter P " _p .

The plasmid pFRBLVsF6 is amplified non-conjuously after addition of chloramphenicol into the medium.

The essence of the construction of the plasmid pFRBLVsF6 is that the fragment of the env gene from the BLV encoding the amino acids 56-103 of the mature gp51 in plasmid pFRBLV-F6 is truncated so that only amino acids 56-64 of mature gp51 including the recognition site for the monoclonal antibody mAK14.

The producer strain of frCP-BLVgp51sh is obtained by transformation of E. coli K802 cells with recombinant plasmid DNA pFRBLVsF6.

Morphological features: gram-negative rod-shaped cells,

Cultural features: Cells grow on common nutrient media, form colonies of medium size,

Physico-biochemical characteristics: optimal cultivation temperature 37 ⁰ C, pH optimum 7.0-7.4. As a carbon source

Hydrocarbons, used as nitrogen source both mineral salts and organic compounds in the form of peptone, tryptone and amino acids.

Antibiotic resistance: resistant to ampicillin due to the presence of the plasmid.

The presence of plasmid DNA in the cell strain is confirmed by control of ampicillin resistance, as well as recovery and analysis of plasmid DNA.

The cell strain is deposited in the collection of the Central Museum of Industrial Microorganisms of the USSR under the number WKPMW4983.

embodiment

The invention will be explained in more detail below using an exemplary embodiment. The accompanying pictures show

Fig. 1: Construction scheme of the recombinant plasmid DNA pFRBLV-F6

Fig. 2: Construction scheme of the recombinant plasmid DNA of the invention pFRBLVsF6.

The realization of the method according to the invention takes place in 3 process steps:

1. Construction of Recombinant Plasmid DNA pFRBLV-F6

20μg of the plasmid DNA containing the BgI Il-Bam HI fragment of the env gene and recovered by standard methods was probed with 50 units of the BglII restriction enzyme and 50 units of the Bam HI rostriktase in 100 μl of solution A (10 mM TrW-HCl , pH 7.5; 50 mM NaCl; 10 mM MgCl ₂ ) were cleaved within 2 hours at 37 ° C. The restriction is inactivated by 15 minutes of heating at 65 ⁰ C. After purification of the Bgl Il-Bam HI-DNA fragments in the agarose gel, by means of known methods, the DNA is dissolved in 20μΙ H] O. The overhanging ends are filled in 100 μl of solution B (50 mM THs-HCl, pH 7.5, 10 mM MgCl ₂ , 10 mM DTT, 25 mM NaCl, 100 μl dATP, dCTP, dTTP, dGTP, 10 units DNA polymerase E. coli / Klenow fragment) within one hour at 12 ⁰ C by. After precipitation with ethanol, the DNA is dissolved in 1OpIH ₂ O (DNAI).

2 pg of the plasmid pFR 20 is cleaved, the 3 units of the Eco RV restriction in 25 μΙ of the solution C (10 mM Tris-HCl, pH 7.5; 5OmM NaCl and 1OmM MgCl ₂ ) within one hour at 37 ⁰ C. The reaction carries onto a 1.5% agarose gel and extract the linearized plasmid DNA (DNA2).

0.1 μg of DNA1 and 0.1 μg of DNA2 are combined by means of DNA ligase in 10 μl of solution D (50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 10 mM DTT, 50 μM ATP and 10 units T ₄ DNA ligase ) within 12 hours at 4 ⁰ C. The enzyme was inactivated by heating at 65 for 5 minutes 1 ^C C.

To the reaction mixture are added ΙΟΟμΙ cells of E. coli RR1 previously treated with 10 μM CaCl ₂ (final concentration of 5 × 10 ⁹ cells / ml) to transfect the recombinant plasmid DNAs.

The clones are selected by sequencing the plasmid DNAs obtained by rapid method. The plasmid with the expected sequence is named pFRBLV-F6 (FIG. 1).

2. Construction of the recombinant plasmid DNA pFRBLVsF6

2pg of plasmid pFRBLV-F6 is cleaved with 3 units 3 units Eco78l and Ssp11 25μΙ in the solution A (10OmM Tris-HCl, pH 7.5, 5OmM NaCl, 1OmM MgCl ₂₎ within one hour at 37 ⁰ C. The inactivated Restrikosen is by a 15 minute heating at 65 ⁰ C. the filling of the protruding ends is carried out in 200μΙ of solution B (5OmM Tris-HCl, pH 7.5; 1OmM MgCl _2; 1OmM DTT; 25 mM NaCl; 100μΜ each dATP, dCTP, dGTP , dTTP, 10 units of E. coli / Klenow fragment DNA polymerase) within one hour at 12 ° C. After purification in an agarose gel, the DNA is dissolved in 20 μM H ₂ O (DNA 3).

0.05 μg of DNA 3 are religated in 20 μl of solution D (50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 10 mM DTT, 50 μM ATP and 10 units of T ₄ DNA ligase) within 4 hours at 4 ° C. , The enzyme was inactivated by a 15 minute heating at 65 ⁰ C.

To the reaction mixture, 100 μl cells of E. coli RR1, previously treated with 10 mM CaCl ₂ (final concentration 5 × 10 ⁹ cells / ml), are added to transform the recombinant plasmid DNAs.

The clones are selected by restriction analysis and sequencing of the plasmid DNAs obtained by rapid method. The plasmid with the expected sequence is named pFRBLVsF6 (FIG. 2).

3. Transformation of the Plasmid According to the Invention Into a Producer Strain The producer strain is obtained by transformation of E. coli K802 cells with the recombinant plasmid pFHBLVsF6.

The cells are multiplied in M9 media with the addition of 10 μg / l casamic acid, 2 g / l glucose and 0.02 g / l ampicillin to an optical density of ODj ₆₀ 4-5.

The characterization of the recombinant protein according to the invention is carried out as follows:

Determination of frCP activity by radial immunodiffusion

The frCP titer is determined by radial immunodiffusion (RID) according to Ouchterlony. This is done by preparing a dilution series (1: 2 ⁿ ; η 1) of the Zeilysat and titrating against rabbit anti-frCP antibodies. As a standard, purified coat protein of phage fr from recombinant bacteria (1 mg / ml) is used. The results of the titration of frCP activity by RID are shown in Table 1.

Synthesis of the recombinant protein frCP-BLVgp51sh in E. coli K802 cells containing the recombinant plasmid DNA pFRBLVsFS

Strain OD ₆₆₀ titer in protein frCP yield of

RID mg / ml mg / ml product (%)

E.COÜK802 4.0 1: 256 20.0 1.0 b, 0

(PFRBLV-F6)

Determination of gp51- and fr-coat protein AktlvitSt by immunoblotting For immunoblotting the cells (6mg) are resuspended in 100μΙ sample buffer according to Laemmli containing 2% SDS and 2% ß-Meracptoäihanol, and lysed by heating in a boiling water bath for 2 minutes. The lysates obtained are applied to a gradient polyacrylamide gel (12-18%) (size 150 mm × 150 mm × 0.75 mm). The electrophoresis is carried out for 15 hours at a current of 7 mA.

After electrophoresis, the proteins are transferred to nitrocellulose HAWP. The filters are treated with the murine monoclonal anti-gp51 antibody mAK14 at a 1: 500 dilution in TBS buffer (50 mM Tris-HCl, pH 7.5, 0.15M NaCl, 0.1% Triton X 100, 1% BSA ) Incubated at 20 ° C for 15 hours. After washing three times in TBS buffer, the filters are incubated for 2 hours at 20 ° C. with the peroxidase-labeled conjugate of the antibodies against mouse immunoglobulin (dilution 1: 500). The filter washes the TBS buffer (3-5χ) and developed with diaminobenzidine.

In Parallelans & tz incubating the filters with ami-FRCP antibodies from the rabbit at a dilution of 1: 200 in TBS buffer After washing twice with TBS buffer is incubated for 15 hours at 2O ⁰ C. the filters for 2 hours at 2O ⁰ C and Peroxidase conjugate of protein A from S. aureus.

The cell lysates of the producer strain in both cases have specific color zones which correspond to a protein of molecular weight 14.7 kD (or a length of 140 amino acids), which was also expected according to the construction scheme (Figure 2). The control rates of E. coli Kd02 cells show no such zones.

Claims (3)

1. A process for the preparation of the recombinant protein frCP-BLVgp51sh, characterized in that in the 2nd amino acid of the coat protein from the phage for the sequence of the epitope BLVgp51 shs, which corresponds to the section between the 56th and 64th amino acid of the mature gp51 incorporated , and the recombinant protein has the antigone properties of both proteins. 2. The method according to claim 1, characterized in that the recombinant plasmid DNA pFRBLVsF6, consisting of the DNA of the vector plasmid pFR 20, which contains the gene bla, which mediates the ampicillin resistance, and the fr-quat protein gene, under the control of a promoter P, _fp , and the fragment of the BLV genome lambda HU1, which corresponds to the sequence between the restriction sites Bgl II and Eco78I with a length of 29 bp and encodes the region of the mature gp51 from the 56th to the 64th amino acid, allows the synthesis of the recombinant protein frCp-BLVgp51sh. 3. The method according to claim 1 and 2, characterized in that one obtains the producer strain of frCP-BLVgp51sh by transformation of E. coli K802 cells with recombinant plasmid DNA pFRBLV-F6.