RU2282661C2 - Recombinant plasmide encoding hybrid gst-esat-6 polypeptide having activities of species-specific mycobacterial esat-6 antigen, recombinant escherichia coli strain as producer of hybrid gst-esat-6 polypeptide and recombinant gst-esat-6 polypeptide - Google Patents

Abstract

FIELD: biotechnology and gene engineering.

SUBSTANCE: invention relates to recombinant plasmide encoding hybrid GST-ESAT-6 polypeptide having activities of species-specific mycobacterial ESAT-6 antigen. Plasmide has molecular mass of 3.45 MD and size of 5315 n.p. Recombinant E.coli polypeptide containing such plasmide and recombinant GST-ESAT-6 polypeptide also are disclosed. Recombinant protein has activities of species-specific antigen protein Mycobacterium tuberculosis ESAT-6. Method of present invention makes in possible to simplify purification process of recombinant polypeptide and to produce protein of high purity hawing activities of mycobacterial ESAT-6 antigen without degradation thereof.

EFFECT: earlier species-specific diagnosis of tuberculosis infection.

3 cl, 3 dwg, 4 tbl, 5 ex

Description

The invention relates to biotechnology, in particular to genetic engineering, and allows microbiological synthesis using the simplified technology to produce a new hybrid GST-ESAT-6 polypeptide with the properties of the species-specific antigen protein Mycobacterium tuberculosis ESAT-6, which can be used for early species-specific diagnosis of tuberculosis infection.

The variety of forms of tuberculosis, the spread of the erased picture of the disease, as well as the individual reaction of the body to the infection are a serious problem for the diagnosis of tuberculosis infection in general and for early diagnosis in particular.

The classical methodology for the diagnosis of tuberculosis is associated with the use of skin (intradermal) reactions to the introduction of tuberculin (PPD), a purified protein derivative [1]. However, the problem of the specificity and sensitivity of skin tests in the case of BCG vaccinated individuals does not distinguish them from individuals infected with pathogenic tuberculosis mycobacteria in this test [2]. One of the reasons for false-positive reactions in the Mantoux reaction is associated with the manifestation of cross-reactivity to non-tuberculous mycobacteria. About 10% of people are reactive to non-tuberculous mycobacteria, of which 30% are positive in a skin test.

Among the classical methods for diagnosing tuberculosis, the method of identifying the culture of the pathogen in the culture of the test sample has the most specificity. Cytological (microscopic) methods for determining mycobacteria in smears after staining with dye were also widely used. However, both of these methods turn out to be of little use in case of nebacillary forms of tuberculosis, when mycobacteria are absent in biological samples. In addition, the cultivation of mycobacteria due to their slow growth is a lengthy procedure, and the cytological identification does not have sufficient sensitivity, is fraught with errors and largely depends on the qualifications of the staff.

Serodiagnostic methods based on the determination of antibodies to mycobacterial antigens are currently widely used for screening studies due to the relatively low cost, speed, high sensitivity and specificity of infected people [3]. Serological methods are very diverse. The sensitivity and specificity of tests using ELISA technology varies over a fairly wide range and amounts to 40-85% and 67-100%, respectively. However, often the indicators of the humoral immune response in tuberculosis are reduced, and this leads to the fact that the virulent mycobacteria that cause tuberculosis in humans cross-react with a set of antigens of non-pathogenic mycobacteria and many pathogens of other diseases. On the other hand, antimycobacterial and hormonal therapy have a suppressive effect on the production of antibodies in tuberculosis. This factor is the main factor in identifying markers of humoral immunity in patients with tuberculosis (antibody levels decrease), and thus, the percentage of positive results in ELISA is relatively low [4].

The literature describes a large number of new proteins (polypeptides) of Mycobacterium tuberculosis complex, which can be used in diagnostic tests. Nucleotide sequences encoding these proteins are also known. Most of this collection resembles other Mycobacterium species and even genera of other bacteria. The amount of culture filtrate proteins secreted by M. tuberculosis mycobacteria and having immunological and enzymatic activity is associated with pathogenesis. However, a complete analysis of the protein composition (composition) in these fractions remains not fully understood [5].

With the development of tuberculosis infection, an important role belongs to T-lymphocytes and other T-cells or T-cell effectors (CD4, CD8) [6]. In particular, CD4-T cells produce immune interferon (gamma-interferon, IFN-γ), which is a stimulator of the production of antimycobacterial macrophages in the infected body, as shown in mouse models [7], as well as an activator of human macrophages in suppressing M. infection. tuberculosis [8].

A comparison of the nature of the change in T-cell and B-cell immunity allows you to identify the level of protective reaction of the body's immune system, aimed at the synthesis of antibodies to bacterial antigens. Studying the state of intracellular functional activity of immunocompetent cells allows obtaining information about the pathological process [9].

A high level of γ-IFN production stimulates T-cell activation, characterizing cellular immunity in tuberculosis invasion. The choice of γ-interferon as a prognostic factor in the diagnosis of tuberculosis is not accidental. Impaired functioning in the IFN chain is a reflection of impaired function of the immune system [10].

γ-IFN analysis is based on a quantitative determination of the level of γ-IFN during stimulation of whole cell blood lymphocytes incubated for 16-20 hours with M. tuberculosis protein antigens and control antigens. As a result of the stimulation of memory T-cell effectors in the blood, the fast secretion of cytokines responsible for the effector functions of the cellular immune response occurs, while IFN-γ is one of the few cytokines that is produced in this process and serves as a specific marker for cellular mediators of the immune response.

As a complex of mycobacterial antigens for such diagnostic studies, tuberculin (PPD) or culture filtrates (CF) of M. tuberculosis H37Rv can be used. In contrast to the skin test, γ-IFN analysis allows differentiation between BCG-vaccinated and infected patients. As a rule, the results of a study of clinical isolates using gamma-interferon analysis reveals a lower percentage of vaccinees who have a positive result for the presence of tuberculosis infection than the data of the intradermal test. This difference indicates that skin test results using PPD have low specificity for BCG-vaccinated patients.

The most suitable polypeptides playing the role of mycobacterial antigens in gamma-interferon analysis are proteins that are highly specific for virulent strains of tuberculosis pathogens and are simultaneously absent in BCG vaccine strains.

The use of species-specific proteins allows us to differentiate different phases of the development of tuberculosis infection in contrast to other M. tuberculosis proteins, for example, the use of a protein with a molecular weight of 19 kDa, and a high level of T-cell response to species-specific antigens correlates with protective immunity in tuberculosis and with the risk of developing an active form tuberculosis.

The ESAT-6 protein is known, first identified by Sorensen et al. [11] as highly specific for virulent species of tuberculosis pathogens. The main advantage of this protein is that there is no nucleotide sequence encoding it in the genomes of the BCG vaccine strains. The molecular weight of the purified native protein is 24 kDa. ESAT-6 is the main antigen causing the development of an early T-cell response. This process is accompanied by increased production of γ-IFN. The ESAT-6 protein gene is part of the 9.5 kbp genomic RD1 fragment present in the genomes of M. tuberculosis and M. bovis strains. But this nucleotide sequence (RD1 region) is absent in all vaccine strains of M. bovis BCG, which means that ESAT-6 is also absent in vaccine preparations, which allows its use for diagnostic purposes [12].

Isolation and purification of ESAT-6 protein from mycobacterial culture filtrate is an expensive and complex procedure, and the product yield is extremely low, therefore, the use of recombinant DNA techniques to obtain this protein is economically more expedient for practical diagnostic purposes.

A known genetic engineering method of producing a recombinant polypeptide with the properties of the mycobacterial antigen ESAT-6, used for diagnostic purposes [13, prototype]. However, the proposed design does not allow to produce preparative amounts of this polypeptide. In addition, during biotechnological purification of the obtained recombinant ESAT-6 polypeptide, the latter undergoes significant degradation, which affects its immunogenic properties.

An object of the invention is the construction of a recombinant strain of bacteria Escherichia coli, a producer of a hybrid polypeptide with the properties of the species-specific mycobacterial antigen ESAT-6, which has such a design that would allow to produce the target highly purified non-degraded polypeptide in preparative quantities while maintaining the immunogenic properties of the latter.

The problem is solved by constructing recombinant plasmid DNA pTSE6 encoding a chimeric polypeptide: glutathione-S-transferase (GST) + ESAT-6 (hereinafter rESAT-6). In this case, the gene encoding the polypeptide with the properties of the ESAT-6 mycobacterial antigen is in the same reading frame with the glutathione S-transferase gene from Schistosoma japonicum (GST S.j.).

Recombinant plasmid DNA pTSE 6 (figa and 1b) with a molecular weight of 3.45 MDa has a size of 5316 bp and consists of the following elements:

- EcoRI-BamHI - fragment of the vector plasmid pGEX-2T (Pharmacia Biotech) 4938 bp in size containing the β-lactamase gene, inducible tac promoter, internal lacI ^q gene encoding the repressor protein of the lactose operon, glutathione-gene gene fragment S-transferases from Schistosoma japonicum with a multiple site for cloning genes at the 3'-terminal portion of this gene and a thrombin proteolysis site;

- EcoRI-BamHI - 378 bp fragment containing the full esat-6 gene flanked by sites for restriction endonucleases EcoRI and BamHI, obtained by amplification of the fragment from the Mycobacterium tuberculosis genome;

contains:

- as a genetic marker, the β-lactamase gene that determines the resistance of E. coli cells transformed with plasmid pTSE6 to the antibiotic ampicillin;

- Unique restriction sites: BamHI - 930, EcoRI-1308.

The presence of a GST fragment in the chimeric recombinant rESAT-6 polypeptide allows the preparation of preparative amounts of the target protein using affinity chromatography. These structural properties are important in the biotechnological aspect. In addition, the presence of the GST fragment makes it possible to facilitate the purification of the recombinant polypeptide and to obtain a highly purified protein with the properties of the ESAT-6 mycobacterial antigen without degradation. The presence of the GST fragment in the composition of the chimeric protein allows you to protect it from intracellular degradation factors and blood enzymes during the stimulation of whole cell blood culture during the diagnostic analysis. The prolongation of its action during the stimulation of whole blood or whole blood mononuclear cells using such structures is one of the important stability factors for such proteins. Moreover, the immunogenic properties of the proposed chimeric structure of the polypeptide are not lost. The fundamental difference between the proposed chimeric polypeptide and the original one in the ESAT-6 prototype is that the recombinant protein obtained as a result of expression is a single entity, including immunodominant species-specific structural epitopes of the native ESAT-6 protein and lacking the functional activity of the GST fragment. The thrombin hydrolysis site located between the GST and ESAT-6 polypeptide fragments (Figs. 2a and 2b) allows, if necessary, the ESAT-6 mycobacterial antigen free of the N-terminal GST polypeptide fragment.

To obtain a bacterial strain producer of the chimeric protein rEsat-6, competent Escherichia coli BL21 cells are transformed with the constructed target plasmid pTSE6. The resulting bacterial strain E.coli BL21 / pTSE6 is characterized by the following features:

Morphological signs. According to morphological characteristics, the producer strain of the recombinant protein rEsat-6 does not differ from the original Escherichia coli BL21 strain that does not contain the target plasmid.

Cultural signs. According to cultural characteristics, the producer strain of the recombinant protein rEsat-6 does not differ from the original Escherichia coli BL21 strain that does not contain the target plasmid.

Antibiotic resistance. The cells of the producer strain of the recombinant rEsat-6 protein exhibit ampicillin resistance due to the presence of the target plasmid.

A significant difference between the E. coli BL21 / pTSE6 strain is that it provides the synthesis of the rESAT-6 chimeric polypeptide (GST-ESAT-6) with the properties of the ESAT-6 mycobacterial antigen with an expression level of 1-10 mg / ml protein.

The resulting strain was deposited at the Research Institute of the Collection of Microorganism Cultures (Research Institute of CMC) of the State Scientific Center of the World Bank "Vector" under the number B-1026.

The chimeric rESAT-6 polypeptide (GST-ESAT-6) (FIGS. 2a and 2b) expressed by the recombinant E. coli strain BL21 / pTSE6 contains the N-terminal polypeptide fragment of glutathione S transferase S.j. as a carrier protein. (226 a.a. with a molecular weight of 26 kDa) and connected via a thrombin hydrolysis site (LVPI ^ GS) C-terminal polypeptide fragment of the species-specific mycobacterial antigen ESAT-6 (95 a.a. with a molecular weight of 6 kDa), and has an amino acid the sequence shown in Fig.2A and 2B.

The invention is illustrated by the following graphic materials.

Figa. The scheme of the organization of the recombinant plasmid pTSE6, where: GST - protein gene of glutathione-8-transferase; ESAT-6 is a cloned fragment comprising the entire gene of the ESAT-6 protein; unique BamHI and EcoRI restriction endonucleases sites used to create this construct; ptac is a synthetic promoter present in vector pGEX-2T and recombinant plasmids; Ap ^R - β-lactamase gene (bla-gene), which provides the transformed bacterial cells with resistance to ampicillin.

Fig. 1b is a restriction map of the target plasmid pTSE6.

Figures 2a and 2b show the complete nucleotide and amino acid sequence of the recombinant GST-ESAT-6 polypeptide, where is the LVPR ^ GS amino acid sequence of the thrombin protease recognition and hydrolysis site (highlighted in color).

For a better understanding of the invention, the following are examples of its specific implementation.

Example 1. Construction of a recombinant plasmid pTSE6.

The genomic mycobacterial DNA of M. tuberculosis strain H37Rv isolated from inactivated mycobacterial cells is used as a source for producing the cloned nucleotide material. The polymerase chain reaction is carried out in a volume of 50 μl in the presence of a pair of primers:

5'-GA GGATCC ATGACAGAGCAGCAGTGG-3 '[direct primer for the esxA gene (esat6)],

5'-GC GAATTC TAAACACGAGAAAGGGCG-3 '[reverse primer for esxA gene (esat6)]

with a temperature-time profile: 94 ° C - 3 min, 4 cycles [94 ° C - 30 s, 45 ° C - 20 s, 72 ° C - 40 s], 33 cycles [94 ° C - 30 s, 58 ° C - 20 s, 72 ° C - 40 s], the resulting annealing of 72 ° C - 5 min. The complete nucleotide sequence of the cloned gene (esxA) for the selection of the primary nucleotide sequence of the primers was obtained from the database of the Pasteur Institute (France). The melting points of the primers based on their nucleotide sequence were calculated using the NTI Suite program. The primers are annealed in the first 4 cycles at a low annealing temperature (45 ° С), calculating the melting temperatures of primers with only fully hybridizing sequences of these primers with a nucleotide genomic matrix: a 5'-terminal sequence of 8 bp is additional and is intended to create a recognition site for restriction endonucleases BamHI and EcoRI, respectively, in order to facilitate the subsequent stage of cloning.

The reaction mixture includes a polymerase buffer containing 60 mM Tris-HCl, pH 8.5; 25 mM KCl; 1.5 mM MgCl ₂ ; 10 mM 2-mercaptoethanol; 0.1% Triton X-100; four deoxynucleoside triphosphates - dATP, dGTF, dTTP and TTF at a final concentration of each of them of 0.2 mm; primers in the concentration of each 0.15-0.2 μm and 1 unit Taq DNA polymerase (Sibenzyme). The yield is about 2 μg (8 pmol) of the PCR fragment. The resulting PCR fragment of 388 bp in length purified by reprecipitation with ethanol in the presence of sodium acetate, pH 4.8, and tRNA as a carrier [14]. The purified amplicon preparation was dissolved in 80 μl of TE buffer and then co-hydrolyzed in EcoRI buffer (Sibenzyme) with restriction endonucleases BamHI and EcoRI at 37 ° C for 3-5 hours. The target restriction obtained from amplicon was reprecipitated with two sticky ends as described above, dissolved in 40 μl of TE buffer or double distilled water and used without further purification in a ligation reaction with a split vector molecule (pGEX-2T), also containing two sticky ends - BamHI and EcoRI. 2-5 μl of the restriction amplicon (0.5 pmol) are mixed with 500 ng (0.15 pmol) of pGEX-2T / BamHI / EcoRI vector DNA in 20 μl of ligase buffer (Sibenzyme), heated at 65 ° C for 3-5 minutes, cool rapidly in ice, add 20 units. Phage T4 DNA ligases and the reaction mixture are transferred to a thermostat, where they are kept at 16 ° C overnight. An aliquot of the ligase mixture (1-2 μl) is used to transform competent E. coli cells of strain JM103 prepared according to the method using a solution of calcium chloride [14], which are seeded on plates with agar medium containing 50 μg / ml ampicillin. The plates are incubated at 37 ° C. overnight. The search for clones containing the desired insertion is carried out directly from the colonies using PCR analysis using oligonucleotides that are complementary to different chains of the vector molecule and located on both sides of the fragment cloning site as amplifiers. Moreover, clones containing the embedding of the complete esxA gene (esat6) after separation of the amplification in agarose gel (1.2%) correspond to a length of 548 bp, which exceeds the length of the cloned fragment, since this fragment contains part of the nucleotide sequence of the original vector molecule . After selection, the target plasmids are produced in analytical quantities and the necessary restriction analysis is performed (detection of the presence of BamHI and EcoRI restriction sites). The resulting target plasmid was designated as pTSE6. Scheme of plasmid DNA is presented in Figa and 1B.

Example 2. Obtaining a producer strain, the cultivation of cells which produces the chimeric protein GST-ESAT6.

Cell transformation of E. coli strain BL21 is carried out by introducing the DNA of the target plasmid pTSE6 into competent cells prepared according to the method using a solution of calcium chloride [14], after which they are plated on plates with agar medium containing ampicillin at a concentration of 50 μg / ml. The plates are incubated at 37 ° C. overnight. The colonies that appeared are clones of the producer strain.

Cells from each selected clone (usually no more than 10) are seeded in 1 ml of liquid medium with ampicillin at a concentration of 50 μg / ml to obtain overnight cell cultures and incubated at 37 ° C without swing. The next day, night culture of each clone is inoculated with a ratio of 1: 100 2 ml of fresh medium with ampicillin at a concentration of 50 μg / ml and incubated at 37 ° C with a swing speed of 160 rpm. The final selection of clones is carried out according to the ability of the cell culture to produce the recombinant protein rESAT6 in the maximum possible amounts after induction, carried out by adding isopropyl-β- to the density of 0.15-0.2 at 660 nm in a growing culture containing cells of each individual clone D-galactopyranoside (IPTG), and subsequent cell cultivation at the selected temperature (in our case, it is 37 ° C and 1 mM IPTG). The concentration of the protein produced by the cells of an individual clone is evaluated after electrophoresis of cell culture lysates in 10% SDS page under denaturing Laemmli conditions.

Example 3. Isolation of the recombinant protein GST-ESAT-6.

Cells of E. coli strain BL21 transformed with pTSE6 plasmid were grown in LB medium with ampicillin (50 μg / ml) overnight at 37 ° C. An overnight culture (1: 100) is seeded in 150 ml of LB medium with ampicillin (50 μg / ml) and grown to the early logarithmic phase. Next, expression of the target GST-ESAT-6 protein is induced by the addition of IPTG to a concentration of 0.5 mM. Induced cells grow for 4 hours at 37 ° C, after which they are harvested by centrifugation at 5000 g. The expression of the target GST-ESAT-6 protein is analyzed by Lammley electrophoresis on a 12% polyacrylamide gel and immunoblotting with monoclonal antibodies to the GST carrier protein. The results of this analysis show the presence of a protein with a molecular weight of about 32 kDa in the induced culture of E. coli BL21 / pTSE6 cells when treating cell pellets with GST-ESAT-6 lysis buffer, which corresponds to the calculated molecular weight. In the control lysates of E. coli BL21 and E. coli BL21 / pGEX-2T cells, there is no protein with this molecular weight. This protein is stained by immunoblotting with monoclonal antibodies to the GST carrier protein.

For further analyzes, the cells are resuspended in 8 ml of cold (0 ° C) PBS buffer containing 140 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ pH 7.3, 1 mm PMSF protease inhibitor, and destroyed by ultrasound on an ultrasonic disintegrator (Ultrasonic processor Cole Farmer) 3 × 30 s with an interval of 1 min between treatments at an amplitude of 50A. Triton X-100 up to 1% is added to the ultrasonic disintegrate, incubated for 30 min at 0 ° C and the fraction of soluble proteins is separated from the debris by centrifugation at 12000 g for 40 min. The supernatant is then mixed with 200 μl of glutathione-sepharose (Glutathione Sepharose 4B Amersham, Biosciences AB) and incubated for 30 min at low stirring speed at room temperature for affinity binding of the GST-ESAT-6 protein. The mixture is then transferred to a microcolumn by repeated centrifugation at 600 g, the column is washed several times with PBS buffer and the target protein is eluted with an elution buffer containing 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8. As a result, a purified recombinant protein preparation is obtained with a concentration of 1 mg / ml.

Example 4. Carrying out IFN-γ analysis.

Stage 1 (stimulation of a whole cell culture of blood with the target antigen). Samples of heparinized venous blood of patients are mixed carefully in a circular motion for 20 minutes. Distribute 100 μl into 96-well plates (Nunc, USA) containing 150 μl / well of complete glutamine RPMI 1640 medium. Thoroughly mix and make antigen GST-ESAT-6 at a concentration of 10 μg / ml per well. Separately, 100 μl of buffer solution are added to wells with zero control (without antigen addition) and PHA mitogen at a concentration of 5 μg / ml per well. Mix gently. Boards are incubated for 20-48 hours at 37 ° C in a humid atmosphere containing 5% CO ₂

2nd stage. To carry out IFN-γ analysis, 100 μl of standard samples of gamma-interferon in different concentrations (from 300-150-75-37.5-18.8-9) are added to the control wells of a plate with sorbed monoclonal antibodies to gamma interferon manufactured by BD Biosciences , 4-4.7 pc / ml) - manufactured by BD Biosciences. 100 μl of the studied blood plasma samples are added to the remaining wells of the tablet after stimulation, placed in a shaker and incubated for 2 hours at room temperature. After incubation, the plate is washed 3 times with wash buffer (FSB-T). After washing, 100 μl of the conjugate of biotinylated anti-IFN-γ antibodies produced by BD Biosciences were added to each well of the plate and incubated for 60 minutes at room temperature. At the end of the incubation, the plate is washed 3 times with FSB-T buffer solution. Then, 100 μl of horseradish avidin peroxidase conjugate manufactured by BD Biosciences in working dilution was added to each well of the plate. Incubated for 60 minutes at room temperature. After incubation, the plate is washed 5 times with wash buffer (FSB-T) and 3 times with H ₂ O distilled. Then, 100 μl of a TMB solution (3.3 ', 5.5'-tetramethylbenzidine) is added to each well of the plate, the plate is placed in a dark place and kept at room temperature for 30 minutes. The reaction is terminated by the addition of 50 μl stop reagent (0.9 N H ₂ SO ₄ solution) to each well of the plate. Optical density is measured on an automatic multiscan spectrophotometer at a wavelength of 450 nm.

To assess the concentration of IFN-γ in the studied samples, a calibration curve is constructed in logarithmic coordinates: the abscissa axis is the IFN-γ concentration (pkg / ml), the ordinate axis is the optical density of the sample. According to the obtained values, a calibration curve is carried out and the quantitative content of IFN-γ in pg / ml or ME / ml in the test and control samples is calculated. The results are calculated from the average OD value in three wells (ρ <0.05). Cutoff values for IFN-γ> 300 pg / ml are considered positive.

Example 5. Mononuclear cells of whole cell blood culture (2 × 10 ⁵ cells / well), isolated according to standard procedures using gradient centrifugation in the presence of Ficoll-Gipak reagents, suspended in 50 μl of complete cell culture medium RPMI 1640 and distributed in 96-well plates (Nunc, MaxiSorb, USA), containing 150 μl / well of the complete medium RPMI 1640 produced by the State Scientific Center of the WB "Vector". Then, in each well of the plate, the chimeric antigen GST-ESAT-6 is added at a concentration of 5-10 μg / ml; mitogen (Con A or PHA) was added to control wells at a concentration of 4 μg / ml in triplicates. The final volume of the reaction medium in the well of the plate is 250 μl. The plates are incubated for 16-24 hours at 37 ° C in a humid atmosphere containing 5% CO ₂ .

To carry out IFN-γ analysis, 100 μl of standard and control samples are introduced into the control wells of a plate with sorbed monoclonal antibodies to gamma interferon. 100 μl of the studied plasma samples after stimulation are added to the remaining wells of the plate. Placed in a shaker and incubated for 2 hours at room temperature. After incubation, the plate is washed 3 times with wash buffer (FSB-T). After washing, 100 μl of the conjugate of biotinylated anti-IFN-γ antibodies are added to each well of the plate and incubated for 60 minutes at room temperature. At the end of the incubation, the plate is washed 3 times with FSB-T buffer solution. Then, 100 μl of horseradish avidin peroxidase conjugate in working dilution was added to each well of the plate and incubated for 60 min at room temperature. After incubation, the plate is washed 5 times with wash buffer (FSB-T) and 3 times with distilled water. Then, 100 μl of a TMB solution (3.3 ', 5.5'-tetramethylbenzidine) is added to each well of the tablet, the tablet is placed in a dark place and kept at room temperature for 30 minutes. The reaction is terminated by the addition of 50 μl stop reagent (0.9 N H ₂ SO ₄ solution) to each well of the plate. The optical density of the samples is measured on an automatic spectrophotometer type "Multiscan" at a wavelength of 450 nm.

To assess the concentration of IFN-γ in the studied samples, a calibration curve is constructed in logarithmic coordinates: the abscissa axis is the IFN-γ concentration (pkg / ml), the ordinate axis is the optical density of the sample. According to the obtained values, a calibration curve is carried out and the quantitative content of IFN-γ in pg / ml or ME / ml in the test and control samples is calculated. The results are calculated from the average OD value in three wells (ρ <0.05). Cutoff values for IFN-γ> 300 pg / ml are considered positive.

Example 6. Sorption of MCA to IFN-γ produced by BD OptEIA Reagent was carried out using 0.1 M carbonate buffer, pH 9.5 for 16 hours at 4 ° C on tablets (Nunc, MaxiSorb, USA). After washing the plate with washing buffer solution (FSB, pH 7.0) 3 times 300 μl / well, the surface of the wells is blocked with a FSB blocking buffer solution, pH 7.0, containing 10% fetal serum, for 60 minutes at room temperature. After that, the tablet is washed, as described above.

A prepared plate with adsorbed MCAs for IFN-γ is used for IFN-γ analysis. To do this, 150 μl of complete RPMI 1640 medium is added to each well of the tablet. Patient heparinized venous blood samples are mixed gently and then dispensed 100 μl into 96-well plates (Nunc, USA). Thoroughly mix and make a solution of GST-ESAT-6 antigen in RPMI 1640 with 0.2 mM glutamine at a concentration of 10 μg / ml per well. Mitogen (PHA) was added to wells A-1, B-1, and C-1, and 100 μl of dilution buffer solution (zero control) was added to wells D-1, E-1. Mix gently. The plates are incubated for 24-48 hours at 37 ° C in a humid atmosphere containing 5% CO ₂ with stirring. After incubation, the plate is washed with 5 × 300 μl / well of distilled water and 5 × 300 μl of PBS-T buffer solution. In order to construct a calibration graph during IFN-γ analysis, incubation is preliminarily performed with standard IFN-γ calibration samples. For this, 100 μl of standard samples of gamma-interferon in different concentrations (from 300-150-75-37.5-18.8-9.4-4) are introduced into the control wells of a plate with sorbed monoclonal antibodies to gamma-interferon manufactured by BD Biosciences , 7 pc / ml) - manufactured by BD Biosciences or 100 μl of standard calibration samples containing IFN-γ (2000-1000-500-300-100-50-0 pcg / ml) and a control sample (330 pc / ml IFN- γ) produced by Vector-Best. Incubate according to the instructions for use of the kits. After incubation, the strips were washed 3 times with wash buffer (PBS-T). After washing, 100 μl of the conjugate of biotinylated anti-IFN-γ antibodies are added to each well of the plate and incubated for 60 minutes at room temperature. At the end of the incubation, the plate is washed 3 times with FSB-T buffer solution, 100 μl of avidin-peroxidase conjugate or horseradish streptavidin-peroxidase in working dilution are added to each well of the plate and incubated for 60 min at room temperature. After incubation, the plate is washed 5 times with wash buffer (FSB-T) and 3 times with distilled water and 100 μl of TMB solution (3.3 ', 5.5'-tetramethylbenzidine) is added to each well of the tablet. The tablet is placed in a dark place and kept at room temperature for 30 minutes. The reaction is terminated by adding 50 μl of a stop reagent (0.9 N H ₂ SO ₄ solution) to each well of the plate and absorbance is measured on a Multiscan type automatic spectrophotometer at a wavelength of 450 nm. The analysis results are presented in Cutoff values, which are obtained from the constructed calibration curves as described above.

Tuberculosis control is carried out to detect active tuberculosis and screening for people who have contact using a tuberculin test. A quick way to detect blood infections early using recombinant proteins is an alternative skin test. Detection of IFN-γ levels correlates with the stage and degree of tuberculosis infection, the level of immune response and the likelihood of progression of the active form of tuberculosis. Table 1 presents the results of the detection of active pulmonary tuberculosis using IFN-γ analysis, as well as among healthy populations. As the studies show, the obtained recombinant proteins allow us to identify the active tuberculosis process among patients with pulmonary tuberculosis. The level of IFN-γ among a healthy population does not exceed the limit values (> 300 pg / ml IFN-γ) corresponding to the risk group or patients with tuberculosis. These data indicate the specificity of the method. The clinical status of patients with various forms of tuberculosis infection identified using rESAT-6 fusion proteins is presented in Table 3. Table 4 presents the results of a comparative study of the specificity and sensitivity of rESAT-6 recombinant proteins compared to PPD-tuberculin in the stimulation of whole cell blood culture . The specificity of recombinant proteins in the IFN-γ analysis of tuberculosis is higher than PPD-tuberculin.

Table 1
IFN-γ - analysis of tuberculosis during stimulation with hybrid proteins in patients with active pulmonary tuberculosis Active tuberculosis patients IFN-γ (pg / ml) RESAT-6 Cona PHA PPD one 1750 6700 4782 4100 2 2360 7324 5421 3650 3 110 4530 3218 2850 four 4475 7300 6710 4870 5 1320 9650 8971 4640 6 27 7861 8764 6200 7 4210 12344 14230 17320 8 760 4851 4352 3980 9 3240 19870 21340 5830 10 3420 18300 17235 10350 eleven 2290 16540 14530 21200 12 1318 12347 13247 5730 13 64 23450 32410 670 fourteen 5640 17650 18930 15340 fifteen 17 640 985 23 16 64 23410 28750 7458 17 3340 19340 18745 10480 eighteen 1680 34500 29560 6130 Donors <300 <300 <300 180-632

Cutoff values for IFN-γ> 300 pg / ml are positive. The results were calculated from the average OD value in three wells (ρ <0.05) and expressed in pg / ml using a calibration graph.

For another group of patients with tuberculosis infection, IFN-γ production results were expressed in IU / ml using a calibration graph. The analytical sensitivity of the test was 1.2-1.7 IU / ml IFN-γ for the negative (zero) control sample from the blood plasma of the studied patients. The results are presented in table 2.

Table 2.
IFN-γ - analysis of pulmonary tuberculosis when stimulated with hybrid proteins and mitogens Patients with active
Form of tuberculosis IFN-γ (IU / ml) PPD rESAT-6 Mitogen PHA TB1 54.3 2,34 63.5 TB2 27.0 3,1 47.6 TB3 59.2 5.7 123.0 TB4 73.0 3.2 117.0 TB5 24.8 2.7 43,2 TB6 11.7 1.73 23.1 TB7 4.7 0.87 126.8 TB8 2.9 0.81 21.3 TB9 5.3 0.73 53.1 TB10 27.6 0.83 143.9 TB11 57.2 0.23 65.3 TB12 10.9 6.4 72.1 TB13 62.3 7.2 57.4 TB14 1.7 2.1 19.7 TB15 33,4 1.7 34.5 TB16 7.2 13.2 67.3 TB17 98.7 12,4 78.1 TB18 74.6 19.3 32.3 TB19 43.1 132.2 179.3 TB20 67.3 147.5 165.7

Table 3.
Clinical status of tuberculosis patients identified using recombinant proteins rESAT-6 and IFN-γ analysis NP / P Tuberculosis infection Sensitivity (%) Specificity (%) one Infiltrative pulmonary tuberculosis 86-95 87-97 2 Focal pulmonary tuberculosis 87-94 93-94 3 Disseminated pulmonary tuberculosis 84-97 86-94 four Fibrous cavernous pulmonary tuberculosis 96-100 97-100 Table 4.
Sensitivity and specificity of PPD, rESAT-6 in TB patients and healthy donors The number of IFN-γ PCG / ml Antigen The number of identified TB patients / to the total number. Sensitivity (%) * Specificity (%) ** TB Healthy *** > 300 PPD 5/6 7/7 83 0 rESA T-6 4/6 4/14 73 71 > 1000 rESA 9/10 3/21 90 85.7 T-6 PPD 7/9 13/14 78.7 7 > 3000 rESA T-6 17/21 2/19 80.9 89.5 * - Positive response among TB-infected patients
** - Positive response among uninfected patients.
*** - BCG-vaccinated and unvaccinated patients

Some forms of tuberculosis (acutely progressive) cannot be detected by the method of preventive examinations. Patients with such processes fall mainly in general hospitals, where their diagnosis can be difficult due to the similarity of the X-ray picture with non-specific lung diseases. The only reliable diagnostic method in such cases is direct sputum bacterioscopy or the use of IFN-γ analysis. All the studied groups of patients with pulmonary tuberculosis had a positive reaction using microscopic methods, culture, fluoroscopy and molecular biological (PCR analysis).

LITERATURE

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Claims (3)

1. Recombinant plasmid DNA pTSE 6 encoding a hybrid GST-ESAT-6 polypeptide with the properties of the species-specific mycobacterial antigen ESAT-6, with a molecular weight of 3.45 MDa, has a size of 5316 bp and consists of the following elements: EcoRI-BamHI - fragment of the vector plasmid pGEX-2T ("Pharmacia Biotech") 4938 bp in size containing the β-lactamase gene, inducible Tac promoter, internal lacI ^q - gene encoding a protein - repressor of the lactose operon, glutathione gene fragment -S-transferases from Schistosoma japonicum (GST) with a multiple site for cloning genes at the 3'-end of this gene and a thrombin proteolysis site in one reading frame; EcoRI - BamHI - 378 bp fragment containing the complete esat-6 gene flanked by restriction sites EcoRI and BamHI, obtained by amplification of a fragment of the Mycobacterium tuberculosis genome; contains: as a genetic marker, the β-lactamase gene that determines the resistance of E. coli cells transformed with plasmid pTSE 6 to the antibiotic ampicillin; unique restriction sites: BamHI - 930, EcoRI - 1308. 2. Recombinant bacterial strain Escherichia coli BL21 / pTSE 6 containing recombinant plasmid DNA pTSE 6 according to claim 1, deposited in the Research Institute of KKM SSC VB "Vector" under number B-1026, producer of the hybrid polypeptide GST-ESAT-6 with the properties of species-specific mycobacterial antigen ESAT-6. 3. Recombinant polypeptide GST-ESAT-6 with the properties of species-specific protein antigen Mycobacterium tuberculosis ESAT-6, which can be used for early species-specific diagnosis of tuberculosis infection in the IFN analysis, expressed by the recombinant strain Escherichia coli BL21 / pTSE6 according to claim 2, contains as a carrier protein, the N-terminal polypeptide fragment of glutathione S transferase Sj (232 a.a. with a molecular weight of 26 kDa) and connected through the proteolysis site of thrombin (LVPR ^ GS) the C-terminal polypeptide fragment of the species-specific mycobacterial antigen ESAT-6 (95 a.a. with a molecular weight of 6 kDa), and has an amino acid the sequence shown in Fig.2.

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