Polypeptid r designed for the detection of antibodies to human immunodeficiency virus the first type, the dna fragment hp 102 encoding the polypeptide r, recombinant plasmid dna ph a102 encoding a polypeptide r, the bacterial strain escherichia coli producing polypeptide r

 

(57) Abstract:

The environment - biotechnology, genetic engineering and immunology. The invention consists in that the hybrid polypeptide R consisting of a polypeptide capable of binding antibodies to the reverse transcriptase product pol gene of HIV-1 and a-galactosidase of E. coli. The hybrid polypeptide is synthesized by bacterial strain Esherichia coli VKPM N B-5876. The strain obtained by transformation of plasmid DNA pH P 102, bearing a fragment of the pol gene of HIV-1.

The invention relates to biotechnology, genetic engineering and immunology and is a hybrid polypeptide consisting of a polypeptide product of a fragment of the pol gene of human immunodeficiency virus 1 type (HIV-1), responsible for the synthesis of the reverse transcriptase of HIV-galactosidase of E. coli synthesized by a bacterial strain of E. coli carrying the recombinant plasmid, having in its composition a cloned fragment of the pol gene of HIV-1 (pol1) fused with the gene b-galactosidase of E. coli and communicating with antibodies to the products of this gene, which allows for serodiagnostic of acquired immunodeficiency syndrome (AIDS).

AIDS is caused by a virus infection, immunodefence the phages and cells of the Central nervous system. The disease is characterized by long-term (from 2 to 10 years) asymptomatic period during which the blood of an infected determined only antibodies to viral proteins, followed by a progressive degradation of the immune and Central nervous system. Therefore, to make an emergency anti-epidemic, preventive measures and early treatment of HIV infection is extremely urgent to develop diagnostic products, allowing to detect HIV in the early stages of infection.

Numerous studies of HIV has allowed to define its structure, genome organization, clarify the mechanism of the expression and function of individual viral proteins. Currently, there are two types of human immunodeficiency virus (HIV-1 and HIV-2), with similar genome organization and different antigenic structure of viral proteins.

HIV is the most complex of all known retroviruses structure of the genome. In addition to the genes gag, pol and env, directing the synthesis of the main virionyx proteins common to all retroviruses, the genome of HIV-1 contains six genes: vif, tat, rev (or art, or trs), 3'-nef, vpr and vpu. The genome of HIV-2, in contrast to HIV-1 does not contain a vpu gene, but contains a gene vpx (Science, 1986, 231, R. (reverse transcriptase), reverse transcriptase is the main protein polymerase complex virus and performs functions associated with the reproduction of genetic information. Although reverse transcriptase - non-structural protein antibodies to it are found in the serum of almost all HIV-positive persons, and the appearance of such antibodies is one of the criteria for full seroconversion. Another important feature of this protein is its relative invariance from isolate to isolate. All it says on the diagnostic importance of HIV reverse transcriptase. It is known that the reverse transcriptase of HIV has three antigennegative determinants are primarily in people living with HIV antibodies are formed (AIDS Research and human retroviruses, 1989, 5, p. 61).

Most of the methods of diagnosis of HIV infection based on the detection of antibodies to HIV proteins in serum and other biological fluids (serodiagnosis). For these purposes, different approaches are used, which applies the principle of enzyme-linked immunosorbent assay (ELISA), agglutination reaction (latex, erythrocytes), immunofluorescence, immunoblotting and radioimmunoprecipitation. One of the most promising ways to improve all of these diagnostic test-systems is used as the antibody-binding substrate recombinant elkow, preserving antigenic determinants of HIV, instead of viral antigens not only increases the specificity and sensitivity of the test systems, but also makes them safe at work. There are a significant number of developments aimed at creating recombinant proteins, gene products of HIV, and in particular the env gene (EPO patents, N 0265 785, C 12 N 15/00, 1986; EPO 0 270 114, With 12 N 15/00, 1986; EPO, 0276 591, 12 N 15/00, 1986 and others).

Currently searching polypeptides of the most promising for the diagnosis, treatment and prevention of AIDS. Research lead in the direction of the selection cell producers (CIS countries, 0265 785, 12 N 15/00, 1986; PCT 89/03878, With 12 N 15/00, 1989) and in the direction of choosing the most antigennegative determinants (CIS countries, NN 0306219 and 0311228, With 12 N 15/00,1986 and 1987, respectively) and the creation of recombinant polypeptides, gene products of HIV (PCT 91/05864, With 12 N 15/48, 1989; EPO, 0361749, With 12 N 15/48, 1989).

The essence of this technical solution is that proposed the original hybrid polypeptide R containing three antigennegative determinants of reverse transcriptase inhibitors and b-galactosidase of E. coli, binding of antibodies to the reverse transcriptase, the product of the pol gene of HIV-1; the DNA fragment encoding the polypeptide R and part ASS="ptx2">

Producing strains characterized by the following characteristics: the cells straight, rod-shaped, motile with peritrichous flagella, gram-negative, risperadone. Cells grow on the LB medium and other nutrient media used for cultivation of Eschrichia coli and simple nutrient media containing 10 μg/ml tetracycline and 50 μg/ml ampicillin. When grown on nutrient agar medium at 37oWith a colony of cells smooth, round, shiny, pale yellow, transparent, smooth edge. When growing on the same media, but when 30-32oWith colonies of cells are "slimy" phenotype, characteristic colonies of cells with lon mutations, growing at low temperature. With the growth in liquid media the cells form a smooth intense haze. Cells grow well at temperatures from 4 to 37oAt optimum pH from 6.8 to 7.5. As the nitrogen source cells use as a mineral salt in ammonium and nitrate form, and organic forms in the form of peptone and amino acids. Reducing nitrates to nitrites. Gelatino not thin. Maltose is not fermented. Urease activity is not formed. Transformation of cells with plasmids with the promoters of bacteriophage l performed by standard methods. Paul is the ia for the recombinant protein is carried out at a temperature 39-42oC. the Productivity of the strain when using plasmid expression vectors with promoters of bacteriophage l is about 500 µg of recombinant protein per 1 ml of cell suspension in culture density of 1 x 108cells/ml Protein is stable at a temperature of 4oWith in the next 3-4 months.

Recombinant polypeptide R, isolated and purified from the producer strain immobilized on a solid medium (PVC or polystyrene tablets, nitrocellulose, nylon, latex, erythrocytes, and others), binds antibodies to the products of the pol gene of HIV-1 in blood serum and other biological fluids. The strain is deposited in the collection of industrial microorganisms Institute of genetics and selection of industrial microorganisms N-5876.

Example 1. Obtaining a DNA fragment NR.

HIV-infected person is withdrawn from the vein in 10 ml of heparinized blood, which is added to 30 ml of lyse solution (155 mM NH4Cl; 10 mM KHCO3; 0.1 mM EDTA). The mixture is incubated for 15 min on ice. Centrifuged at 2000 G for 10 min at 4oC. White cells resuspended in 10 ml of SE (75 mM NaCl, 2 mM EDTA) add proteinase K To a final concentration of 100 μg/ml and 1 ml of 20% SDS. The mixture is incubated for 4 h at 37oadd 1/2 volume (5 is ahavat on the rocking chair 15 min and centrifuged at 2000 rpm for 10 minutes For selected aqueous phase add 1/30 volume of NaOAc (pH 5.5) and equal volume of isopropyl alcohol. DNA is then precipitated by centrifugation and washed with 70% ethanol solution. The precipitated DNA was dissolved in buffer TE (10mM Tris-HCl, 1mM EDTA, pH 8.0) to a final concentration of 1 µg/ml.

In eppendorff tube 0.5 ml incorporate the following components: deionized water to 43.5 ál reaction buffer 10 ál x 10 (final concentration 25 mM Tris-HCl, pH 8.3 (at 25oC), 50 mM KCl, 2mM MgCl2, 1 mM b-mercaptoethanol, gelatin, 200 μg/ml), a mixture of dNTP 16 μl (final concentration 200 μm), primer N1 5' TGTACTGATCCCGGATCC 3' 5 μl (final concentration of 1.0 μm), primer N2 5' AGCCTCAGGACCTTGCC 3' 5 μl (final concentration of 1.0 μm), primer N3 5' GCAAAGCAGCCTCAGGACC 3', 5 μl (final concentration of 1.0 μm), primer N4 5' CTGCAGTGGTACCCATGCC 3', DNA template for amplification) 10 μl (final concentration 1 ng), Tag polymerase of 0.5 ál (2.5 units per sample).

The contents of the tubes are mixed on the vortex 3-4 with and precipitate on microcentrifuge for 5-10 sec.

In a test tube add 50 ál of mineral oil and placed in the apparatus for amplifying DNA N 801-0177 DNA Thermal Cycler Perkin-Elmer Corporation.

The reaction mixture was incubated at 94oWith 7 minutes, and then spend the cycle amplification with SL min at 94oC (denaturation of DNA). The loop amplification is repeated 25 times. After the last cycle of amplification, the reaction mixture was incubated at 72oWith 10 minutes

In test tube add 50 μl of chloroform, the material is shaken with chloroform with 5 on the vortex and then centrifuged at microcentrifuge 10 C. Select the aqueous (upper) phase (about 100 µl), which is formed in the form of spherical droplets, and transferred to a clean tube.

10 µl of the aqueous phase used for the analysis of amplification products in 2% agarose gel with methyl-ethidium (you can see two fragment sizes 600 and 170 p. N.). Synthesized on a DNA array DNA fragments hydrolyzing the restriction enzyme MstII in Bromphenol blue buffer (10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl and 1 mM dithiothreitol pH 7.5) for 14 h at a temperature of 37oC. the Enzyme inaktiverad by heating at 70oWith 15 minutes DNA precipitated with ethanol and dissolved in 10 μl of N2O. 10 μl of treated restrictable DNA fragment is placed in eppendorff tube containing 3 μl of ligase buffer (50 mM Tris-HCl, 10 mM MgCl2, 20 mM dithiothreitol, 1.0 mM ATP, 50 μg/ml bovine serum albumin), 1 μl (100 units) of T4 DNA ligase and 16 μl of N2O. the Mixture is incubated for 3 h at 16oC. as a result of ligation of polycavernoside DNA fragment NR.

GGATCCGGGATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCC

AAAGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAACAAAATCCAGACATAGTTATCT

ATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAA

AAATAGAGGAGCTGAGACAACATCTGTTGAGGTGGGGACTTACCACACCAGACAAGAAAC

ATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAG

TACAGCCTGTAGTACTGCCAGAAAAGGACAGCTGGACTGTCAATGACATACAGAAGTTAG

TGGGGAAATTGAATTGGGCAAGTCAGATTTACCCAGGGATTAAAGTAAGGCAATTATGTA

AACTCCTTAGAGGAACCAAAGCACTAACAGAAGTAGTACCATTAACAGAAGAAGCAGAGC

TAGAACTGGCAGAAAACAGAGAGATTCTAAAAGAACCAGTACATGGGGTGTATTATGACC

CATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGTCCTGAGGCTGCTTTGC

AGGATTCAGGATTAGAAGTAAACATAATAACGGACTCACAATATGCATTAGGAATCATTC

AAGCACAACCAGATAAAAGTGAATCAGAGTTAGTCAATCAAATAATAGAGCAGTTAATAA

AAAAGGAAAAGGTCTATCTGGCATGGGTACCACTGCAG

Example 2. Obtaining plasmids RNR.

Synthesized on a DNA array DNA fragment hydrolyzing restrictase BamH1 and Pst1 in the Bromphenol blue buffer (10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl and mM dithiothreitol pH 7.5) for 14 h at a temperature of 37oC. the Enzymes are inactivated by heating at 70oWith 15 minutes DNA precipitated with ethanol and dissolved in 50 μl of H2O. 10 MLK treated restrictable DNA fragment is placed in eppendorff tube containing 3 μl of ligase buffer (50 mM Tris-HCl, 10 mM MgCl2, 20 mM dithiothreitol, 1.0 mM ATP, 50 μg/ml bovine serum albumin), 2 μl (0.2 ág/ml) DNA vector plasmid pEL5B treated restrictase BamH1 and Pst1 and 4 μl of H2O. To this mixture 1 μl (100 u) 14 the Tamm PLT90 by conventional methods (Molecular cloning. New York, CSHL, 1982). Transformed cells are plated on plates with 1% LB:agar containing 25 μg/ml of ampicillin. Grown colonies screened for recombinant plasmids. For this purpose, the bacterial cells are lysed and release of plasmid DNA, as described (Nature, 1981, 145, 1365). The selected plasmid DNA is treated with restriction enzyme BamH1 and Pst1 and hydrolyzed examined by electrophoresis in 1% agarose.

The hydrolysate of plasmid DNA from recombinant strain HP102 has on the electrophoresis strip 2 DNA size 6400 and 770 nucleotides that correspond to the dimensions of the vector and we synthesized DNA fragment. When determining the nucleotide sequence of the plasmid, named RNR, it was found that the synthesized our DNA fragment NR via BamH1 site is connected to the 3'end of the gene b-galactosidase of E. coli.

Example 3. The strain of E. coli NR containing plasmid RNR, grown in 100 ml LB medium at 30oWith up to a density of 8 x 10 cells/nl. After that, the temperature was raised to 37oWith and grow the cells for 2 hours, the Cells are harvested by centrifugation at 4000 rpm for 15 minutes, the Cells are lysed by ultrasound and secrete proteins according to the described method (EMBO J. 1984, 3, 1429).

Selected proteins analyzed in 10% SDS page on a standard methodsi markers molecular masses ranging from 20 to 160 KD. As a control using the obtained similarly the cell lysate of E. coli strain PLT90 containing the vector plasmid L5B and not containing the obtained plasmid RNR. Comparative analysis of protein composition shows that the strain of E. coli HP102 containing plasmid RNR, expresses a hybrid polypeptide with a molecular mass of 140 to 150 KD. This polypeptide is named R. Amino acid sequence of the hybrid polypeptide (sequence b-galactosidase not shown) defined on the basis of the nucleotide sequence of the cloned fragment of proviral DNA pol gene of HIV-1 is presented below:

IRDQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYVGSDLEIGQHRTK

IEELRQHLLRWGLTTPDKKHQKEPPFLWMGYELHPDKWTVQPVVLPEKDSWTVNDIQKLV

GKLNWASQIYPGIKVRQLCKLLRGTKALTEVVPLTEEAELELAENREILKPVHGVYYDP

SKDLIAEIQKQGQGPEAALQDSGLEVNIITDSQYALGIIQAQPDKSESELVNQIIEQLIK

KEKVYLAWVP

fused with b-galactosidase of E. coli.

Example 4. Control of antigenic activity.

Control antigenic activity of preparations carried out by the method of immunoblotting. To do this, hold the procedure of electrophoresis (as described in example 3), then carry out the electrophoretic transfer of separated proteins to nitrocellulose membrane VN using the apparatus for electroplating mode 24V, 400mA in eczematous temperature. The paint washed twice for 30 min with TBS buffer (0.5 M NaCl1, 20 mM Tris-HCl, pH 7.4) with 0.05% Tween-20 at room temperature. In the next step, conduct blocking nitrocellulose membranes with immobilized protein a solution of 5% nonfat dry milk prepared in 0.1 M Na-phosphate buffer, pH 7.4, for 30 min at room temperature. After blocking the membrane for 1 h at 37oWith treated with serum containing antibodies to HIV-1, dilution 1:100. Then spend three times washing of the membranes with TBS of 0.05% Tween-20 for 30 min at room temperature. Detection of specific antibodies is performed by incubation of the filter with antivitamin peroxidase conjugate at 37oC for 1 h Then repeat the procedure three times of washing. The reaction is checked by adding 100 ál of 30% hydrogen peroxide and 50 ml of a solution of 4-chloro-1-naphthol (20 mg in 0.1 M Tris-Hcl, pH 8.0). The analysis shows that the polypeptide R associated with antibodies to the human immunodeficiency virus the first type.

Thus, this invention allows to identify antibodies to the products of the pol gene of HIV-1 with high efficiency and specificity, and the process definition does not require working with wisecontact is immunodeficita person of the first type, obtained in the bacterial strain Escherichia coli VKPM N-5876, transformed with recombinant plasmid RNR that encodes a polypeptide with the following amino acid sequence:

< / BR>
merged with beta-galactosidase of E. coli, and having the ability to bind with antibodies to the reverse transcriptase product pol gene of HIV-1 mol. m 140 150 kilodaltons when determining the method of polyacrylamide gel electrophoresis.

2. The DNA fragment HP 102 encoding the polypeptide P 102 and received by chain reaction polymerization of the nucleotide sequence of

< / BR>
containing the 3'end of the stop codon.

3. Recombinant plasmid DNA PHP 102 encoding a polypeptide P 102 intended for the detection of antibodies to human immunodeficiency virus first type, size 7170 p. O. containing BamHI Pst DNA fragment of the vector pL5 containing the gene for beta-galactosidase of E. coli size 6400 p. O. > PST Bam HI DNA fragment HP 102 encoding the polypeptide P 102 size 770 p. O. one plot splitting restrictase BamHI and Pst I; the promoter of bacteriophage lambda cro LacZ; genetic markers: AmpRthe gene for resistance to ampicillin.

4. The bacterial strain Escherichia coli VKPM N-5876 producer polypeptide P 102 intended d

 

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