RU2089612C1 - Recombinant plasmid dna purvgf determining synthesis of smallpox virus strain l-ivp growth factor together with escherichia coli beta-galactosidase and strain of bacterium escherichia coli containing recombinant plasmid dna purvgf - a producer of smallpox virus strain l-ivp growth factor - Google Patents

Abstract

FIELD: biotechnology, molecular biology, genetic engineering. SUBSTANCE: invention relates to construction of recombinant plasmid DNA determining smallpox virus growth factor synthesis and preparing the strain containing the indicated plasmid. Strain is used as a producer of smallpox virus growth factor fused with β-galactosidase. Recombinant smallpox virus growth factor can be used in future as mitogen for epithelial cells, for cell proliferation and wound healing. EFFECT: plasmid indicated above. 2 cl, 3 dwg

Description

 The invention relates to biotechnology and medicine, in particular to biologically active substances and methods for their preparation by genetic engineering.

As it became known, the vaccinia virus growth factor (VGF) is assigned to the family of EGF-like factors [1] The mature VGF molecule has 35% amino acid homology with epidermal growth factor (EGF) and binds to EGF receptors on the cell surface, causing cell proliferation [2]
Under normal conditions, VGF is produced by the vaccinia virus when it is reproduced in cell cultures, and the amount of this protein in the culture medium is extremely small, and the method of its purification from the culture medium is complicated and requires expensive equipment and reagents.

 The creation of a bacterial producer of VGF will allow it to be obtained in preparative quantities and purified to a homogeneous state using a relatively simple purification technique.

The literature describes the VGF gene of the vaccinia virus strain WR virus, as well as the positive effect of this factor isolated from the culture medium of cells infected with the vaccinia virus or synthesized chemically on cell proliferation in vitro [4.6]
Also known is a gene coding for the composition of the Hind III G-DNA fragment of the vaccine vaccinia virus L-IVP strain of vaccinia virus vaccinia virus growth factor [3,5] The VGF gene is isolated from the clonal variant (clone 4) of the vaccinia virus L-IVP strain different from the VGF gene WR described in the literature [4] by four nucleotide substitutions at positions 86, 319, 358, 359. which leads to amino acid substitutions: at position 29 of serine per leucine, at position 107 methionine per valine and at position 120 leucine per serine [5, prototype]
However, information regarding the creation of recombinant VGF producers suitable for the production of this protein in preparative quantities is not available.

 The aim of the invention is the creation of a recombinant bacterial strain of E. coli-producer VGF vaccinia strain L-IVP clone 4 in the form of a chimeric protein.

The goal is achieved by constructing recombinant plasmid DNA pUR VGF encoding the synthesis of VGF, which has a size of 5.419 N. p. and consists of the following elements (Fig. 1):
of the vector part in the form of Hind III BamH I fragment of plasmid pUR 290 with a size of 5.186 bp containing the LacZ gene with its own promoter Ibla-gene.

BamHI Hind III fragment, 233 bp containing the sequence of the open translation frame (ORT) of the VGF virus vaccinia virus strain L-IVP gene without sequences encoding the signal and transmembrane parts of the VGF gene (Fig. 3)
The pUR VGF plasmid DNA contains unique restriction sites for the BamHI, Hind III, Tag I, Ava II, Alu I, Nla III, Cla I enzymes.

 To achieve this goal, a method for constructing the pUR VGF plasmid is used, namely, the vector part obtained by cleavage of the plasmid DNA pUR 290 with BamH I Hind III restriction endonucleases is fused using phage T4 DNA ligase with a DNA fragment of the vaccinia virus treated with the same endonucleases which has been previously amplified by the polymerase chain reaction method using pre-synthesized primers complementary to the terminal sequences of the smallpox vaccine virus DNA region, Odir mature VGF. In addition, a site encoding an acid labile bond and a stop codon are introduced at the beginning of the VGF gene, and restriction sites for Hind III BamH I endonucleases are also introduced.

 A significant advantage of the proposed method is that with its help the expression of the virus-specific VGF protein in E. coli cells is achieved. The VGF protein gene is in the same reading frame with the LacZ gene, which ensures the synthesis of a chimeric polypeptide with mol. m. 126 kDa, consisting of beta-galactosidase and protein VGF, connected acid-labile bond.

 Plasmid pUR VGF is not described in the literature and was first obtained in this work.

 To obtain a producer strain of E. coli, competent cells of strain BMH7118 are transformed with a recombinant DNA plasmid. The producer strain BMH7118 differs from the initial one only in the characters transmitted by the plasmid, i.e. It is resistant to ampicillin and synthesizes a chimeric protein of 126 kDa, consisting of beta-galactosidase E. coli and protein VGF and with beta-galactosidase activity. The yield of VGF is 6 mg / g of bacterial cells. The resulting bacterial producer strain is registered in the All-Union Collection of Industrial Microorganisms of the All-Russian Research Institute of Genetics under the number VKP / M V-6566.

 The resulting strain is characterized by the following features.

1. Morphological features
Cells are straight, rod-shaped, 1.2x1.6x2.0-6.0 μm, motile (with peritrichous flagella), gram-negative, non-spore.

2. Cultural traits
Cells grow well on simple nutrient media, the optimum growth temperature is 30-37 ^o C, pH 6.8-7.5.

 With growth on meat and peptone and fish agar they develop in the form of smooth, round, shiny, gray, cloudy colonies with smooth edges. When working in liquid media cause uniform turbidity with the formation of sediment.

3. Physiological and biological characteristics
The optimum cultivation temperature is 37 ^o C, the optimum pH is 6.8-7.5. As a carbon source, many carbohydrates, alcohols, and organic acids are used. As a source of nitrogen, both mineral salts in ammonium or nitrate forms, as well as organic compounds peptone, amino acids, are used.

4. Resistance to antibiotics
It shows resistance to ampicillin, due to the presence of plasmid pUR VGF at a concentration of the latter 70 to 100 mg / L.

5. Stability of the plasmid in the strain
When maintaining the cells of the strain for 1.5 years on fish agar at +40 ^o C or in 30% glycerol at -70 ^o C in the presence of ampicillin, no rearrangement or loss of plasmid DNA is observed.

 The essence of the invention lies in the fact that as a result of cloning of the VGF gene of the vaccinia virus of the E. coli strain BHM 7118 with the recombinant plasmid pUR VGF DNA, the producer strain BHP pUR VGF is obtained, in which the chimeric polypeptide 126 kDa (beta-galactosidase-VGF) is produced reacting with monospecific serum to the synthesized peptide corresponding to 20-33 amino acid residues of the N-terminus of the mature VGF molecule (Fig. 2).

 In FIG. 1 shows a physical map of plasmid pUR VGF; in FIG. 2 - immunoblotting of the lysate of cells of E. coli strain BMH 7118 after three-hour induction of IPTG (isopropyl-thio-beta-D-galactopyranoside) with monospecific rabbit antiserum obtained to the synthesized peptide (20-33 amino acid residues of the N-terminus of mature VGF).

Tracks:
1 proteins of E. coli cells transformed with pUR VGF plasmid, separated by electrophoresis in 8% PAG;
2 proteins of pUR 290 cells transformed with pUR 290 plasmid, separated by electrophoresis in 8% PAG.

 In FIG. 3 shows the nucleotide sequence encoding mature VGF.

 Example 1. Construction of recombinant plasmid DNA pUR VGF.

В последовательности олигонуклеотидов заложены сайты для рестриктаз BamHI и HindIII, а также стоп-кодон.Using synthetic oligonucleotides primers for polymerase chain reaction (PCR) and viral DNA (strain L-IVP), a DNA fragment is obtained that encodes mature VGF. The parameters of the full-sized chain reaction are as follows: annealing - 37 ^o , 1 min; elongation 72 ^o , 1 min; denaturation 95 ^o , 1 min; number of cycles 25; enzyme Tth polymerase. The structure of the oligonucleotide primers:

The sequence of oligonucleotides contains restriction enzyme sites BamHI and HindIII, as well as a stop codon.

After amplification, 5 μg of DNA fragment with mobility in PAAG about 230 bp is obtained. The indicated DNA fragment is treated with BamHI and HindIII enzymes under standard conditions (50 mM NaCl, 10 mMx2 mercaptoethanol, 10 mM MgCl ₂ , 10 mM Tris HCl, pH 7.5); the reaction is stopped by adding 0.5 m EDTA (5 μl per 100 μl of the mixture), restriction enzymes are removed with phenol and DNA is precipitated from the aqueous phase as described in [7]
The vector is prepared as follows: 5 μg of pUR 290 DNA is cut under standard conditions with BamHI and HindIII restriction enzymes according to the method described in [7] and 5% PAAG is applied. A strip of DNA was isolated and dissolved in 50 μl of TE.

 Vector DNA (0.05 μg) and a fragment (1 μg) were crosslinked with T4 DNA ligase [7]. Recombinant clones were selected by restriction analysis. Competent cells are prepared from E. coli strain JM109.

 Example 2. Obtaining a producer strain of E. coli BMH 7118.

 Bacterial E. coli JM109 cells containing pUR VGF plasmid DNA were grown in 4 ml of L-broth with ampicillin at a concentration of 40 μg / ml to a titer of 5x108 cells / ml. Plasmid DNA was isolated by the alkaline method [3] and retransformed into E. coli cells of strain BMH 7118. The resulting strain was analyzed for the presence of plasmid markers.

 1. Resistance to ampicillin.

 Cells of E. coli BMH7118 strain are plated on an agar medium with an antibiotic content of 10 to 100 μg / ml. The growth of these cells is not inhibited to a concentration of 100 μg / ml.

 2. Expression of the chimeric polypeptide of 126 kDa.

Cells of E. coli strain BMH7118 induce IPT G (IOE-3 M) and lysed in 3% SDS (50 mM 2-mercaptoethanol; 20 mM Tris-HCl pH 6.8; 30% sucrose 5 ml) at 95 ^° C. Separated denatured polypeptides disk electrophoresis in 8% PAG and detected using Coomassie R-250. The molecular weight of the chimeric polypeptide of 126 kDa is calculated by electrophoretic mobility.

 3. The enzymatic activity of the chimeric polypeptide of 126 kDa.

 The cells of the strain are seeded on an agar medium containing 40 μg / ml ampicillin, 0.1 mg / ml X-gal, 0.1 μg / ml IPT G. Colonies of E. coli strain JM109 are blue.

 4. The formation of the AG-AT complex of the chimeric protein 126 kDa with a monospecific antiserum to the synthesized peptide (20-33 amino acid residues of the mature VGF molecule) immunogenic properties of the chimeric protein 126 kDa are confirmed in the immunoblotting reaction (Fig. 2), as described below.

 Example 3. Immunochemical analysis of the chimeric polypeptide of 126 kDa.

The bacterial strain of E. coli BMH pUR VGF grow up to D550 0.6 p.u. and after the addition of IPT G, up to 50 μg / ml was grown for 3 hours at 37 ^° C, stirring vigorously. 1 ml of cell culture is centrifuged for 3 min at 6000 pellet suspended in 100 μl of 3% SDS; 5% mM 2-mercaptoethanol; 20 mM Tris-HCl pH 6.8; 30% sucrose and boiled for 1 min; voiced; 1/10 of the resulting solution was separated in 8% PAG and polypeptides were transferred to nitrocellulose. The fixed protein preparation is incubated in a 1% BSA solution, then incubated at room temperature with a monospecific rabbit antiserum against the synthesized peptide for 1 h in 150 mM NaCl; 20 mM Tris-HCl, pH 7.7; 0.2% Triton X-100. Washed three times in a buffer of this composition and incubated for 1 h with a peroxidase conjugate of protein A. After washing in the buffer, the AG-AT complex was stained with 3,3-diaminobenzeneidine HCl.

 The invention allows to obtain VGF in milligram quantities and to study its immunological and biological properties.

 The resulting producer of recombinant VGF can be used to study the interaction of this growth factor with membrane receptors, to produce antibodies to VGF, and in the future it is possible to use recombinant VGF obtained in E. coli cells as a mitogen that causes proliferation of cells of epithelial origin in vivo and in vitro.

 Thus, as a result of the studies, a producer strain E. coli expressing the vaccinia virus growth factor was obtained. Further studies of this protein will create the basis for its use in the treatment of burn disease and for the healing of difficult to heal trophic ulcers and wounds.

Literature
Stroobant R. Rico AP Gullick WJ et al, Cell, 1985, V. 42, P. 383-393.

 Schulz G. White M. Mitchell N. Science. 1987, V. 235, N4786, P. 80-82.

 Petrov V.S. Khromykh A.A. Dmitriev I.P. et al. Genetic and cellular engineering in solving fundamental problems of biotechnology: Materials of the All-Union Conf. Tartu, 1989, vol. I, p. 171-174.

 Venkatesan S. Gershowitz A. Moss B.J. of Virology, 1982, V.44, P. 637-646.

 Petrov V.S. Cheshenko N.V. Belavin P.A. et al. Biotechnology, 1992, N5, p. 35-39.

 Lin J. Caporaso C. Ke X. H. Tam. J. of Biol. Chemistry, 1990, V. 265, N.31, P. 1881-1890.

 Maniatis T. Fritsch Z. Sambrook J. Methods of genetic engineering. Molecular Cloning Per. from English M. World, 1984.

 Salganik R.I. Methods of molecular genetics and genetic engineering. Novosibirsk "Science" 1990.

Claims (2)

 1. Recombinant plasmid DNA pURVGF, which determines the synthesis of growth factor virus smallpox vaccine strain L-IVP together with beta-galactosidase Escherichia coli, size 5419 bp containing Bam HI Hind III DNA fragment of the plasmid pUR 290 size 5186 bp corresponding to the gene for resistance to ampicillin and the beta-galactosidase gene, the Bam HI Hind III 233 bp fragment of the vaccinia virus genome comprising a smallpox vaccine virus growth factor gene region encoding a mature polypeptide, one Bam HI restriction enzyme cleavage site, one Tag I restriction enzyme cleavage site, located at 17 bp from the Bam HI site, one Alu I restriction enzyme cleavage site located at 64 bp from the Bam HI site, one Ava II restriction enzyme cleavage site located at 79 bp from the Bam HI site, two cleavage sites for restriction enzyme Nla III, located at a distance of 160 p. and 187 bp from the Bam HI site, one cleavage site for restriction enzyme Hind III, located at a distance of 245 BP from the Bam HI site, one cleavage site for the restriction enzyme Cla I, located at a distance of 246 p. from the Bam HI site, genetic marker: beta-lactamase gene that provides ampicillin resistance.  2. The bacterial strain Escherichia coli VKPM B 6566 containing the recombinant plasmid DNA pURV6 F is a producer of the growth factor virus vaccinia strain L-IVP.

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