RU2081171C1 - Recombinant plasmid dna pgdn, method of preparing recombinant plasmid dna pgdn and strain of bacterium escherichia coli containing recombinant plasmid dna pgdn used for preparing the hybrid protein consisting of 579 amino acids and showing antigenic properties of human virus t-cellular leukosis the first type - Google Patents

Abstract

FIELD: biotechnology, genetic engineering. SUBSTANCE: invention relates to methods of detection of human virus T-cellular leukosis the first type (HTLV-1). Method involves preparing recombinant plasmid DNA providing high level of hybrid gag-protein HTLV-1 production showing antigenic property and preparing the strain-producer of the indicated protein. Plasmid pGdN is obtained by fusion of plasmid pVR-290 fragment encoding E. coli β-galactosidase, two polylinkers, start replication region and gene determining the resistance to ampicillin with plasmid MT2 fragment containing the sequence of gag-protein HTLV-1. E. coli cells were transformed with the obtained plasmid and the strain GKV/pGdN is selected. This strain produces hybrid protein containing virus-specific sequence of gene gag HTLV-1. After the strain GKV/pGdN culturing the yield of protein showing antigenic properties of HTLV-1 is 400-500 mg/1 l cell suspension. EFFECT: improved method of plasmid preparing, enhanced effectiveness of strain-producer. 4 cl, 3 dwg

Description

 The invention relates to the field of biotechnology and is a strain of Escherichia coli containing recombinant plasma DNA with fragments of the gag region of the HTLV-1 genome, which determines the synthesis of a hybrid protein having the antigenic properties of HTLV-1, and a method for creating such a plasmid.

 T-cell leukemia virus type I (human T-cell leukemia virus type 1, HTLV-1) is the etiological agent of adult T-cell leukemia / lymphoma of a serious disease characterized by malignant proliferation of OCT-4 + lymphocytes. This virus is also associated with a number of neurological disorders, namely with various forms of myelopathy and tropical spastic paraparesis. The latency period for HTLV-1 infection in some cases can reach 20-40 years. The gag region of HTLV-1 is localized in the region of 824-2113 nucleotide pairs (bp) of the genome and encodes the core proteins of the virus, matrix and nucleocapsid p19, p24 and p15, respectively.

 Recombinant DNA containing a fragment of the gag region encoding an incomplete precursor of the gag gene, and providing synthesis in bacteria Escherichia coli (E. coli) of a hybrid protein, the N-terminal part of which is represented by beta-galactosidase, and the C-terminal virus-specific sequence is not described in the literature .

 The proposed invention allows by creating a recombinant plasmid DNA that provides a high level of production of a hybrid protein possessing the antigenic properties of HTLV-1 and capable of being inherited in E. coli bacterial strains, thereby creating a producer strain of this protein.

Description of the plasmid:
name pGdN;
size 5.9 kb

unique restriction sites BamHI, PstI, StuI, Tth111-II; consists of an NcoI-BamHI fragment of plasmid pUR290 with a size of 5.2 kb with the lacZ gene encoding a full-length beta-galactosidase of 341 ac; two polylinker fragments encoding 4 ak; the ori region and the Ap / r ampicillin resistance gene (FIG. 1);
consists of the NcoI SmaI fragment (699 bp) of the plasmid pMT2 (carrying the full-length HTLV-1 genome) containing the sequence of the HTLV-1 gag region and encoding 234 ak of the GAG residue of the HTLV-1 precursor (144 to 377), namely 200 C-terminal ac p24 of the core virion core protein and 34 N terminal amino acids of the p15 protein;
the plasmid determines the resistance of ampicillin to E. coli K12 HB101 cells and the synthesis of a 579 amino acid residue fusion protein containing antigenic determinants of the gag region HTLV-1;
the plasmid is non-conjugative.

 Description of E. coli K12 HB101 / pGdN strain.

 The bacterial strain Escherichia coli K12 HB101 / pGdH used to obtain a hybrid protein with antigenic properties of the human T-cell leukemia virus type 1 was deposited in the Museum of strains and plasmids of the Institute of Virology. D. I. Ivanovsky RAMS. The strain was assigned inventory number STB / pGdH. Strain genotype: F, ehd A1, hsdR17 (r, m), supE44, thi-1, recA1, gyrA96, relA1, f (argF-1ac z, y, a), U169, 0 80d, 1acZ fm15.

 The level of synthesis of the hybrid protein is at least 30 of the total cellular protein and allows you to get 400-500 mg of protein with the antigenic properties of HTLV-1 with 1 liter of cell suspension.

 Morphological signs: cells are straight, rod-shaped 1.2x1.6x2.0 microns, motile with peritrichous flagella, gram-negative, spore-forming.

Cultural traits: cells grow well on simple nutrient media. For the cultivation of E. coli bacteria, a culture medium (L-broth) of the following composition (g / l) was used: bactotriptone or bactopeptone (Difco) -10, yeast extract (Difco) 5, NaCl-10. The solid nutrient medium was LB supplemented with 1.5% agar (Difco). Ampicillin (Ap) resistant clones of E. coli were selected on solid nutrient medium supplemented with 30-100 μg per ml ampicillin. Before use, the nutrient medium was autoclaved for 30 minutes at 120 ^° C. and 1.2 atm. When growing on solid media, colonies are smooth, round, pressed, shiny, gray, cloudy, the edge is even. When growing on liquid media, they form a uniform intense turbidity.

The number of viable cells was determined by counting the colonies grown on solid nutrient medium after 16-18 hours of incubation at 37 ^° C. Suspension of bacterial cells was plated from various dilutions in a volume of 0.1-0.2 ml by grinding with a spatula on the surface of the agar plate. The cell concentration in the bacterial suspension was also determined by the nephelometric method. The optical density was measured on a spectrophotometer at a light wavelength of 590 nm with a light path length of 1 cm. The concentration of viable cells was determined in this case from a calibration curve.

Physico-biochemical characteristics: cells grow in the range from 4 to 45 ^o With an optimum pH of 6.8 to 7.5. Glucose and fructose are used as a carbon source. Acetate is not absorbed, and nitrates are reduced to nitrites. Gelatin is not liquefied; indole is not formed. Urease activity is not detected.

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

 Example 1. Construction of the plasmid pGdN.

 To obtain the first intermediate plasmid pSI, a SmaI fragment (870-1952 bp) was isolated from the previously obtained plasmid pEHgagI (Questions of Virology, 1991, pp. 325-326) containing the Eco47.3 HindIII fragment of the DNA copy of the viral genome in the pUC18 vector, which was used to clone the gag gene (1080 bp) into the pUC8 vector by the same restriction enzyme.

For this, 20 μg of DNA of the plasmid pEHgag1 was incubated with 30 units. SmaI in a buffer containing 33 mM Tris-acetate pH 7.9, 10 mM Mg-acetate, 66 mM K-acetate, 0.5 mM DTT, (buffer "A" from Boehringer mannheim (Germany) for 1 h at 37 ^o C. The reaction mixture was applied to a 1% agarose gel containing ethidium bromide at a concentration of (0.5 μg / ml) and electrophoresis was carried out in Tris-acetate (TAE) buffer for 0.5 h at 100 V. Then, the desired DNA fragment using the Gene Clean kit from B10-101 (USA) by cutting the desired fragment under soft ultraviolet (366 nm) and dissolving it in a 2-3-fold volume of NaJ (solution from the kit) when heated to 45- 50 ^o C. The melt 5 μg of a suspension of glass beads capable of immobilizing DNA was added while 5 minutes later, the beads were besieged on an Eppendorf centrifuge (Germany) for 2 minutes at 10,000 rpm, after which the supernatant was removed. Precipitated glass beads were resuspended in New solution (supplied in a set) on 96% ethanol using a vortex.The procedure is repeated three times, after which the plasmid DNA is washed off the glass beads with distilled water in the required volume by resuspension on the vortex and pellet beads on an Eppendorf centrifuge (Germany). The supernatant contains the desired DNA.

 Two μg of the vector pUC8 were incubated with 30 units. SmaI under the same conditions.

DNA fragments of plasmid pUC8 (0.5 μg) and SmaI fragment HTLV-1 from plasmid pEHgag1 were treated with T4 DNA ligase (3000 u / mg) from Boehringer mannheim (Germany) at the rate of 1 μl of the enzyme per 0.5 mcg of DNA. The composition of the ten-fold ligase buffer: Tris-HCl 660 mM, MgCI 2.50 mM, DTE 10 mM, ATP 10 mM, pH 7.5. Incubation was carried out for 10 hours at 12 ^o C.

The resulting preparation was used to transform competent E. coli HB101 cells obtained by the calcium chloride method. Transformed cells were grown for 1.5 hours and plated on agar medium containing L-broth with ampicillin. Plasmid DNA isolated from ampicillin-resistant colonies grown after 18 hours at 37 ^° C was analyzed by electrophoresis in 1% agarose with the addition of 0.5 μg / ml ethidium bromide. Clones were selected in which the molecular weight of the fusion proteins is greater than the mass of the vector part of the original clone (vector pUC8). The resulting plasmid, in which the orientation of the gag HTLV-1 gene coincided with the reading frame of the beta-galactosidase gene, which was determined by DNA hydrolysis with NcoI and BamHI enzymes, was designated as pSI.

For hydrolysis, 10 μl of plasmid DNA was taken, which was incubated with 20 units. each of the enzymes in the presence of Boehringer mannheim buffer B (Germany) containing 10 mM Tris-HCl pH 8.0, 5.0 mM MgCl-2, 100 mM NaCl and 1.0 mM mercaptoethanol, over 1 h at 37 ^o C.

E. coli HB101 bacterial cells containing pSI plasmid DNA were grown in 200 ml of broth to a titer of 1000 million cells / ml. Cells were pelleted by centrifugation (5000 g, 5 min, 4 ^° C.) and resuspended in 35 ml of a solution containing 8% sucrose, 0.5% Triton X100, 50 mM EDTA pH 8.0 and 10 mM Tris-HCl pH 8.0. Then 2.5 ml of a freshly prepared solution with a concentration of 10 mg / ml was added, quickly mixed and heated in a boiling water bath for 3 minutes. The resulting lysate was centrifuged (20,000 g, 30 min, 4 ^° C) and DNA from the supernatant was precipitated with an equal volume of isopropanol. The precipitate formed was collected by centrifugation (12000 g, 20 min, 2 ^° C) and resuspended in 5 ml of 10 mM Tris-HCl buffer, pH 8.0.

The synthesis of the intermediate plasmid pN1 in direct orientation with the built-in HindIII linker at the EcoRI site of the vector polylinker was carried out as follows: 10 μl of the pSI plasmid was hydrolyzed with EcoRI enzyme in the presence of Boehringer mannheim buffer (H) consisting of 50 mM Tris-HCl with pH 7.5, 10 mM MgCI-2, 100 mM NaCl and 1.0 mM DTE, for 1 h at 37 ^° C. Annealing of 20 μl of the synthesized HindIII linker represented by the ten-member (CCAAGCTTGG) was carried out in a water bath at a temperature of 68 ^o C for 5 min with further cooling in a disconnected bath to room temperature. To ligate the obtained fragments, the enzyme hydrolyzed EcoRI with the enzyme pSI plasmid was treated with 0.5 μl of the Klenov fragment and 5 μl of dNTP's at room temperature for 0.5 h. The ligation process itself was carried out according to the method described above. The ligation material was transformed into a competent culture of E. coli HB101. Colonies grown on agarose medium in the presence of ampicillin were seeded in L-broth with the same concentration of antibiotic with further DNA isolation, as already described.

 The creation of the third intermediate plasmid pGdP was carried out by co-hydrolysis of the BamHI + HindIII plasmid pN1 and the vector pUR 290 by the same restriction enzymes, while maintaining the reading frame and direct orientation of the gag gene.

 The final plasmid pGdN was obtained by co-hydrolysis of pGdP with NcoI + BamHI enzymes and further ligation of the mixture in the presence of a Klenov fragment and dNTP's while maintaining the reading frame of the cloned gene and beta-galactosidase. The resulting mixture was used for transformation. It should be noted that all copies of the plasmid pGdN, after their transformation in E. coli bacteria, synthesizing a hybrid protein with an estimated molecular weight of 145 kDa are identical and are considered as the claimed strain after testing the recombinant polypeptide for the ability to specifically interact with HTLV-1 positive sera in the immunoblot.

Plasmid DNA isolated from ampicillin-resistant colonies grown after 18 hours at 37 ^° C was analyzed by electrophoresis. Clones were selected whose molecular weight was greater than that of the original vector pUR 290.

 To select the desired plasmid, an analysis was made of the ability of the selected plasmids to synthesize hybrid proteins with a molecular weight of 145 kDa containing virus-specific sequences of the gag region.

 Example 2. Analysis of the synthesis level of a hybrid protein with antigenic properties of E. coli HB101 / pGdN.

 The analysis of proteins in E. coli HB101 cell lysates containing the hybrid plasmid pGdN was carried out as follows. Two ml of a suspension of E. coli HB101 cells containing the plasmid pGdN, grown overnight from a separate colony to a concentration of 2000 million cells / ml in L-broth containing ampicillin, precipitated by centrifugation (12000g, 1 min) and resuspended in 200 μl of buffer containing 50 mM Tris HCl 7.8, 2.5% sodium dodecyl sulfate, 10% glycerol, 0.7% mercaptoethanol and 0.1% bromophenol blue. The resulting preparations were boiled for 5 min in a water bath with subsequent analysis by electrophoresis. In the lysates of bacteria containing the plasmid pGdN, an additional protein with a molecular weight of 145 kDa was detected (Fig. 2, a). The antigenic activity of a protein with a molecular weight of 145 kDa was analyzed using the immunoblot method (Fig.2, b). The resulting hybrid protein possessed the antigenic activity of HTLV-1. The level of synthesis of the hybrid protein was at least 30% of the total cellular protein.

 The invention allows to obtain from 1 l of a cell suspension of 400-500 mg of protein with antigenic properties of HTLV-1. In this case, 579 ak of the hybrid protein are presented as follows: 341 ak of beta-galactosidase, 2 ak are encoded by the polylinker sequence of the vector plasmid pUR 290, 234 ak are a fragment of the gag HTLV-1 gene and 2 ak are encoded by the polylinker pUR 290 (terminator), which is shown in figure 3.

 Example 3. Demonstration of the possibility of using the target product to create a diagnosticum on HTLV-1.

 Protein preparations prepared according to the method described in Example 2 were separated by PAGE using electrophoresis and analyzed by immunoblot using the serum of a patient infected with HTLV-1. In the preparation of proteins obtained from cells containing the hybrid plasmid pGdN, additional bands of proteins that specifically react with antibodies to HTLV-1 were detected. In the preparation used as a control, prepared from cells containing the original vector pUR 290, such bands were not identified.

 Thus, the plasmid pGdN determines the synthesis of proteins with the antigenic properties of HTLV-1 proteins in E. coli cells; therefore, this plasmid and producer strain can be used to create diagnosticums for the detection of antibodies to HTLV-1.

Claims (3)

 1. Recombinant plasmid DNA pGdN size 5.9 TPN determining the synthesis of a hybrid protein consisting of 579 amino acid residues (a.a.), possessing the antigenic properties of the human first type T-cell leukemia virus (HTLV-1), containing unique restriction sites for the endonucleases BamHI, PstI, StuI, Tth, 111 II ; a genetic marker that provides ampicillin resistance; 5.2 bp NcoI BamHI fragment of plasmid pUR 290 coding including full-sized beta-galactosidase Escherichia coli and sections of polylinker regions; NcoI-SmaI fragment of plasmid pM T2 0.7 kbp coding sequence of the gag protein of HTLV-I size 234 a.k.  2. A method for producing recombinant plasmid DNA pGdN of size 5.9, etc. n determining the synthesis of a hybrid protein consisting of 579 a.k. possessing antigenic properties of the human first type T-cell leukemia virus (HTLV-I), namely, that the SmaI fragment of the plasmid pEH gag I is inserted into the pUC8 vector at the SmaI site to obtain the pSI plasmid, Hind III is inserted into the indicated plasmid at the EcoRI site linker; the obtained plasmid pNI and the vector pUR 290 are hydrolyzed together with restriction enzymes BamHI and Hind III, the fragments obtained are ligated to form an intermediate plasmid pGdP, in which the reading frame and direct orientation of the gag HTLV-I gene are preserved, then using restriction enzymes NcoI and BamHI from the indicated plasmid pGdP 0.7 kb DNA fragment re-ligated using the Klenov fragment and deoxynucleotide triphosphates while maintaining the reading frame of the cloned gag gene HTLV-I and beta-galactosidase Escherichia coli and obtain the target recombinant plasmid DNA pGdN.  3. The bacterial strain Escherichia coli BKB / pGdN containing recombinant plasmid DNA PGdN, used to obtain a hybrid protein consisting of 579 a.k. possessing antigenic properties of the virus of human T-cell leukemia of the first type.

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