Recombinant plasmid dna pgdn, a method of obtaining a recombinant plasmid dna pgdn and the bacterial strain escherichia coli containing a recombinant plasmid dna pgdn used to obtain a hybrid protein consisting of 579 a. k., possessing antigenic properties of the virus t-cell leukemia person of the first type

 

(57) Abstract:

Usage: biotechnology, development of diagnostic kits for the detection of virus T-cell leukemia person of the first type (HTLV-1). The inventive preparation of recombinant plasmid DNA, providing a high level of production of hybrid gag-protein NTLV-1, possessing antigenic properties and strain-producer of the specified protein. Plasmid pGdN receive when connecting fragment of plasmid pUR 290 encoding beta-galactosidase of E. coli, two polylinker, the starting area of replication and the gene of resistance to ampicillin, with a fragment of the plasmid MT2 containing the sequence of gag-protein of HTLV-I. the Obtained plasmid transformed E. coli cells and selected strain GKV/pGdN, producing a hybrid protein containing virusspecific sequence of the gag gene HTLV-1. Under cultivation of strain GKV/pGdN from 1 l of cell suspension get 400-500 mg protein having antigenic properties of HTLV-1. 3 C. p. F.-ly, 3 ill.

The invention relates to the field of biotechnology and is a strain of Escherichia coli containing recombinant plasma DNA fragments of the gag region of the genome of HTLV-1 that specifies the synthesis of a hybrid protein having antigenic properties of HTLV-1, tsya etiological agent of T-cell leukemia/lymphoma adult severe disease, characterized by malignant proliferation of OCT-4+ lymphocytes. This virus is also associated with a number of neurological disorders, namely various forms of myelopathy and tropical spastic paraparesis. The duration of the latent period of the infection HTLV-1 in some cases can reach 20-40 years. The gag region HTLV-1 is localized in the area 824-2113 base pairs (p. N.) genome and encodes the core proteins of the virus, matrix and nucleocapsid P19, P24 and p respectively.

Recombinant DNA containing the fragment of the gag region encoding an incomplete predecessor of the gag gene, and for synthesis in the bacteria Escherichia coli (E. coli) hybrid protein, N-terminal part of which presents beta-galactosidase, and C-terminal virusspecific sequence, are not described in literature.

The proposed invention allows the creation of recombinant plasmid DNA, providing a high level of production of a hybrid protein having antigenic properties of HTLV-1 and can be inherited in strains of E. coli bacteria, to create thus producing strains of this protein.

Description plasmids:

the title page;

size of 5.9 T. p. N.

the unique restriction sites BamHI, ETA-galactosidase size 341 AK; two fragments of polylinker coding 4 AK; ori region and the gene for resistance to ampicillin Ap/r (Fig.1);

consists of NcoI SmaI fragment (699 p. N.) plasmid pMT2 (carrying a full-sized genome of HTLV-1), containing the sequence of the gag region HTLV-1 and coding 234 AK residue GAG precursor HTLV-1 (144 377), namely 200 C-terminal AK P24 core protein core of the virion and the 34 N-terminal amino acids of the protein R;

plasmid determines the resistance of cells of E. coli K12 HB101 to ampicillin and synthesis of a hybrid protein size 579 amino acid residues containing the antigenic determinant region of gag HTLV-1;

plasmid conjugation.

Description strain of E. coli K12 HB101/pGdN.

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

The level of synthesis of a hybrid protein is at least 30 from the total cell protein and allows you to get from 1 l of cell suspension 400-500 mg of protein, the possession,2x1,6x2,0 μm, motile with peritrichous flagella, gram-negative, paraneoplasia.

Cultural characteristics: the cells grow well on simple nutrient media. For the cultivation of E. coli bacteria used nutrient medium (L-broth) of the following composition (g/l): lactotropes or bactopeptone (Difco)-10, yeast extract (Difco) 5, NaCl-10. Solid nutrient medium served as a LB with the addition of 1, 5% agar (Difco). The selection of clones of E. coli resistant to ampicillin (AP), was carried out on solid nutrient medium with the addition of 30-100 μg per ml ampicillin. Before use, the medium autoclaved for 30 min at 120oC and 1.2 ATM. During growth on solid media of the colony is smooth, round, appressed, shiny, grey, cloudy, smooth edge. With the growth in liquid media to form a smooth intense haze.

The number of viable cells was determined by counting the colonies grown on solid nutrient medium after 16-18 h of incubation at 37oC. Suspension of bacterial cells were sown from different dilutions in the volume of 0.1-0.2 ml by rubbing with a spatula on the surface of the plates with agar. The concentration of bacterial cells in the suspension was determined values method. The optical density measured on sintered who ranged in this case, the calibration curve.

Physical and biochemical characteristics: the cells grow in the range of 4 to 45oAt optimum pH from 6.8 to 7.5. As the carbon source used glucose and fructose. Acetate is not digested, and reducing nitrates to nitrites. Gelatino not thin, do not form indole. Breazna activity is not detected.

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

Example 1. Construction of plasmids pGdN.

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

For this 20 μg DNA plasmid pEHgag1 incubated with 30 units of SmaI in buffer containing 33 mm Tris - acetate pH 7.9, 10 mM Mg-acetate, 66 mm K-acetate, 0.5 mm DTT (buffer, "" manufactured by Boehringer mannheim (Germany) for 1 h at 37oC. the Reaction mixture was applied to 1% agarose gel containing ethidium bromide at a concentration (0.5 μg/ml), and electrophoresis was performed in Tris-acetate (TAE) buffer for 0.5 h at 100 C. it was Further identified need is in the summer (366 nm) and dissolving it in 2-3 times the volume of NaJ (solution set) when heated to 45-50oC. In the molten gel was added 5 g of a suspension of glass beads, can mobilitat DNA. After 5 min the beads were besieged in the centrifuge "Eppendorf (Germany) for 2 min at 10,000 rpm, after which the supernatant was removed. Besieged glass beads resuspendable solution "New" (supplied in kit) in 96% ethanol using a vortex. The procedure is repeated three times, after which the plasmid DNA is washed off with glass beads with distilled water in the required amount by resuspendable on the vortex and deposition of balls in the centrifuge "Eppendorf (Germany). The supernatant contains the desired DNA.

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

The DNA fragments of the plasmid pUC8 (0.5 μg) and SmaI fragment of HTLV-1 from plasmids pEHgag1, and treated with DNA ligase of phage T4 (3000 units/mg), manufactured by Boehringer mannheim (Germany) at the rate of 1 ál of the enzyme at 0.5 μg DNA. The composition tenfold ligase buffer: Tris-HCl 660 mM MgCI 2,50 mM, 10 mM DTE, 10 mM ATP, pH 7.5. Incubation was carried out for 10 h at 12oC.

The resulting preparation was used to transform competent cells of E. coli HB101, potassium chloride obtained by the method. The transformed cells were pokasivali 1.5 h and were sown on agar medium containing L-broth with ampicill the Wali by electrophoresis in 1% agarose with the addition of 0.5 μg/ml ethidium bromide. Were selected clones, in which the molecular weight of the hybrid 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 gene HTLV-1 coincided with the reading frame of the gene beta-galactosidase, which was determined by hydrolysis of DNA by enzymes NcoI and BamHI, was designated as pSI.

For hydrolysis took 10 ál of plasmid DNA that was incubated with 20 units of each enzyme in the presence of buffer "B" manufactured by Boehringer mannheim (Germany), containing 10 mm Tris-HCl c pH 8.0, 5.0 mM MgCl-2, 100 mM NaCl and 1.0 mm mercaptoethanol, for 1 h at 37oC.

Cells of E. coli HB101 containing plasmid DNA pSI, were grown in 200 ml of broth until the titer of more than 1,000 million cells/ml Cells was besieged by centrifugation (5000 g, 5 min, 4oC) and resuspendable in 35 ml of a solution containing 8% sucrose, 0.5% of Triton X100, 50 mm EDTA pH 8.0 and 10 mm Tris-HCl pH 8.0. Next was added 2.5 ml of freshly prepared solution with a concentration of 10 mg/ml, quickly mixed and heated in a boiling water bath for 3 minutes the resulting lysate was centrifuged (20000 g, 30 min, 4oC) and DNA from the supernatant was besieged by an equal volume of isopropanol. The precipitate was collected by centrifugation (12000 g, 20 min, 2oC) and resuspendable in 5 ml of 10 m the dIII site EcoRI polylinker vector was carried out as follows: 10 ál of plasmid pSI hydrolysable enzyme EcoRI in the presence of buffer "H" manufactured by Boehringer mannheim (Germany), 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 37oC. Annealing 20 µl synthetic HindIII linker represented by desyatichenko (CCAAGCTTGG), conducted in a water bath at a temperature of 68oC for 5 min with subsequent cooling in a disabled bath to room temperature. For ligation of the resulting fragments gidrolizirovanny EcoRI enzyme plasmid pSI for 0.5 h, treated with 0.5 μl of fragment maple and 5 ál dNTP's at room temperature. The process of ligation was performed according to the methodology described above. Legirovannye material transformed into competent culture of E. coli HB101. Grown on agarose environment colonies in the presence of ampicillin was grown in L-broth with the same concentration of the antibiotic with a further DNA isolation, as has already been described.

The creation of a third intermediate plasmids pGdP conducted by joint hydrolysis BamHI+HindIII plasmid pN1 and vector pUR 290 for the same restrictases with preservation of the reading frame and direct the orientation of the gag gene.

The final plasmid pGdN was obtained using Conjoint hydrolysis pGdP enzymes NcoI+BamHI and subsequent ligation mixture in the presence of a fragment of maple and dNTP's with preservation of the reading frame of cosmetici, that all copies of the plasmid pGdN, after their transformation into the E. coli carrying out the synthesis of a hybrid protein with an estimated molecular mass of 145 kDa are identical and considered as the claimed strain after the test recombinant polypeptide on the ability to interact specifically with HTLV-1 positive sera in immunoblot.

Plasmid DNA isolated from ampicillin-resistant colonies that grew after 18 h at 37oC, analyzed by electrophoresis. Were selected clones, the molecular weight of which was greater than that of the original vector pUR 290.

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

Example 2. The analysis of the level of synthesis of a hybrid protein having antigenic properties of E. coli HB101/pGdN.

Analysis of proteins in cell lysates of E. coli HB101 containing the hybrid plasmid pGdN, was carried out as follows. Two ml of cell suspension of E. coli HB101 containing plasmid pGdN grown overnight from a single colony to a concentration of 2000 million cells/ml in L-broth containing ampicillin, besieged cent is sodium, 10% glycerol, 0.7% mercaptoethanol and 0.1% bromophenol blue. The resultant preparations were boiled 5 min in a water bath, followed by analysis by electrophoresis. In lysates of bacteria containing the plasmid pGdN found additional protein with a molecular mass of 145 kDa (Fig.2, a). Antigenic activity of a protein with a molecular mass of 145 kDa was analyzed using immunoblot method (Fig.2, b). The resulting hybrid protein possessed antigenic activity of HTLV-1. The level of synthesis of the hybrid protein was at least 30% of the total cell protein.

The invention can be obtained from 1 l of cell suspension 400-500 mg protein having antigenic properties of HTLV-1. However 579 AK hybrid protein is as follows: 341 AK beta-galactosidase, 2 AK encoded polylinker sequence vector plasmids pUR 290, 234 AK represent a fragment of the gag gene HTLV-1 and 2 AK encoded by polylinkers pUR 290 (terminator), which is shown in Fig.3.

Example 3. To demonstrate the feasibility of the use of the target product to create diagnosticum for HTLV-1.

Preparations of proteins prepared according to the method described in example 2 were separated by electrophoresis in SDS page and analyzed metodologia hybrid plasmid pGdN, revealed additional bands of proteins, specifically reactive with antibodies to HTLV-1. The drug, used as a control prepared from cells containing the source vector pUR 290, such lines are not identified.

Thus, plasmid pGdN determines in E. coli cells the synthesis of proteins with antigenic properties of proteins of HTLV-1, therefore, this plasmid and producing strains can be used to create diagnostic kits for detection of antibodies to HTLV-1.

1. Recombinant plasmid DNA pGdN size of 5.9 T. p. N. determining the synthesis of a hybrid protein consisting of 579 amino acid residues.K.), possessing antigenic properties of the virus T-cell leukemia person of the first type (HTLV-1), containing unique restriction sites for the endonucleases BamHI, PST, StuI, Tth, 111 II; genetic marker providing resistance to ampicillin; NcoI BamHI fragment of plasmid pUR 290 size 5,2, etc., ad encoding including a full-sized beta-galactosidase of Escherichia coli and plots polylinker areas; NcoI - SmaI fragment of the plasmid pM T2-0.7, etc. ad coding sequence of the gag protein of HTLV-I size 234.to.

2. A method of obtaining a recombinant plasmid DNA pGdN size of 5.9 T. p. N. definition the human leukemia of the first type (HTLV-I), namely, that SmaI fragment of plasmid pEH I gag is inserted into the vector pUC8 at the SmaI site to obtain plasmid pSI, indicated plasmid at the EcoRI site embed Hind III linker; the resulting plasmid pNI and vector pUR 290 together hydrolyzing restrictase BamHI and Hind III, the resulting fragments are ligated with the formation of intermediate plasmids pGdP, in which the stored frame is read and the direct orientation of the gag gene HTLV-I, next, using restricted NcoI and BamHI from the specified plasmids pGdP cut out a DNA fragment with a size of 0.7 T. p. N. again are ligated using fragment maple and deoxynucleotidase with preservation of the reading frame cloned gene gag HTLV-I and beta-galactosidase of Escherichia coli and receive targeted recombinant plasmid DNA pGdN.

3. The bacterial strain Escherichia coli STB/pGdN containing recombinant plasmid DNA PGdN used to obtain a hybrid protein consisting of 579 A. to. possessing antigenic properties of the virus T-cell leukemia person of the first type.

 

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