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

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

 

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

The variety of forms of tuberculosis, the distribution of erased picture of the disease, as well as individual reaction of an organism to infection represent a serious problem for the diagnosis of tuberculosis infection in General and for early diagnosis in particular.

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

Among the classical methods of diagnosis t the tuberculosis highest specificity has a method of identifying the culture of the pathogen in the crop sample. Widespread also Cytology (microscopic) methods for the detection of tubercle bacilli in smears after coloring dye. However, both of these methods are of little use when abacilar forms of tuberculosis when bacilli are absent in the bioassay. In addition, the cultivation of mycobacteria due to their slow growth is a long process, and cytological identification does not have sufficient sensitivity, error-prone and largely depends on the qualification of personnel.

Methods serodiagnosis based on the detection of antibodies to antigens of mycobacteria that are currently widely used for screening due to the relatively low cost, rapidity, high sensitivity and specificity among infected individuals [3]. Serological methods are very diverse. The sensitivity and specificity of tests using ELISA technology varies in a rather wide range and is 40-85% and 67-100%, respectively. However, often the results humoral immune response in tuberculosis is reduced, and this leads to the fact that virulent mycobacteria that cause tuberculosis in humans, cross-react with a set of non-pathogenic antigens of mycobacteria and many other pathogens B. the diseases. On the other hand, antimycobacterial and hormonal therapy have a suppressive effect on the production of antibodies in tuberculosis. This factor is fundamental in identifying markers of humoral immunity in TB patients (see a lower level of antibodies), and thus, the percentage of positive results in the ELISA is relatively low [4].

The literature describes a large number of new proteins (polypeptides) of the Mycobacterium tuberculosis complex, which can be used in diagnostic tests. Known and nucleotide sequences encoding these proteins. Most of this collection has similarities with other species of Mycobacterium and even other genera of bacteria. The number of proteins in the culture filtrate, secreted by mycobacteria M. tuberculosis and has immunological and enzymatic activity associated with pathogenesis. However, a full analysis of protein composition (composition) in these fractions remains to be elucidated [5].

With the development of TB infection is an important role for T-cells and other T cells or T-cell effector (CD4, CD8) [6]. In particular, CD4-T-cells produce immune interferon (IFN-γ, IFN-γ), which is a stimulator in the infected organism antimycobacterial Mac is ofago, as shown in mouse models [7], as well as an activator of macrophages person in the suppression of M.tuberculosis infection [8].

Mapping changes in T-cell and b-cell immunity helps to identify the level of protective response of the immune system, aimed at the synthesis of antibodies to bacterial antigens. The study of intracellular functional activity of immunocompetent cells allows to obtain information about the pathological process [9].

A high level of product γ-IFN stimulates T-cell activation, characterizing cellular immunity in tuberculosis invasion. Choice as a prognostic factor in the diagnosis of tuberculosis γ-interferon is not random. Malfunction in a chain of the IFN is a reflection of the dysfunction of the immune system [10].

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

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

The most suitable polypeptides that play the role of mycobacterial antigens in gamma interferon analysis, are proteins, highly specific for virulent strains of tuberculosis and at the same time missing in vaccine BCG strains.

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

Known protein ESAT-6, first identified Sorensen and co-authors [11] as it is highly specific for virulent species of causative agents of tuberculosis. The main advantage of this protein in the fact that in the genome of BCG vaccine strains lacking the nucleotide sequence encoding it. The molecular mass of the purified native protein is 24 kDa. ESAT-6 is the major antigen that causes the development of early T-cell response. This process is accompanied by increased production γ-IFN. Gene protein ESAT-6 is part of the genomic fragment RD1 size 9.5 thousand gel present in the genomes of strains of M. tuberculosis and M. bovis. But this nucleotide sequence (RD1-region) is missing in all vaccine strains of M. bovis BCG, and this means that ESAT-6 is also absent in vaccine preparations that can be used in diagnostic purposes [12].

Isolation and purification of protein ESAT-6 from the culture filtrate of Mycobacterium is an expensive and complicated procedure, the yield is extremely low, so the technique of recombinant DNA to obtain the protein is much more economically feasible for practical diagnostic purposes.

Known genetic engineering method of obtaining a recombinant polypeptide with properties of mycobacterial antigen ESAT-6, BL is used for diagnostic purposes [13, the prototype]. However, the proposed design does not allow to develop preparative quantities of the polypeptide. In addition, in the process of biotechnological purification the recombinant polypeptide ESAT-6, the latter undergoes significant degradation, which affects its immunogenic properties.

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

The problem is solved by constructing recombinant plasmid DNA pTSE6 that encodes a chimeric polypeptide: glutathione-S-transferase (GST)+ESAT-6 (hereinafter referred to rESAT-6). When this gene that encodes a polypeptide with properties of mycobacterial antigen ESAT-6, is in the same reading frame with the gene glutathione S-transferase from Schistosoma japonicum (GST unlimited company.).

Recombinant plasmid DNA pTSE 6 (figa and 1B) with a molecular weight of 3.45 MDA has a size 5316 P.N. and consists of the following elements:

- EcoRI-BamHI fragment of the vector plasmid pGEX-2T (Pharmacia Biotech) size 4938 gel containing gene β-lactamase induced ac-promoter, internal lacIq-the gene encoding the protein is a repressor of Lac operon, a gene fragment glutathione-S-transferase from Schistosoma japonicum with multiple site for cloning genes in the 3'end of this gene and the site of proteolysis by thrombin;

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

contains:

as a genetic marker gene β-lactamase determining the stability of the transformed plasmid pTSE6 of E. coli cells to the antibiotic ampicillin;

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

The presence of chimeric recombinant polypeptide rESAT-6 GST-fragment allows to develop preparative quantities of the target protein using affinity chromatography. These design features are important in biotechnological aspect. Furthermore, the presence of GST-fragment allows to reduce the purification of the recombinant polypeptide and to obtain a highly purified protein with properties of mycobacterial antigen ESAT-6 without degradation. The presence of GST-fragment in the composition of the chimeric protein can protect him from intracellular degradation factors and enzymes blood upon stimulation of whole cell cultures of blood in the process diagnostic anal is for. The prolongation of its action upon stimulation of whole blood or mononuclear cells from whole blood with the use of such structures refers to the important factors of the stability of these proteins. This immunogenic properties of the proposed chimeric structure of the polypeptide is not lost. The principal difference of the chimeric polypeptide from the original prototype of the ESAT-6 is that the recombinant protein, the resulting expression is a single education, including species-specific immunodominant structural epitopes of the native protein ESAT-6 and not having a functional activity of GST-fragment. Located between the polypeptide fragments of GST and ESAT-6 (figa and 2B) website hydrolysis by thrombin allows you to get mycobacterial antigen ESAT-6-free N-terminal polypeptide fragment of the GST.

To obtain a bacterial strain-producer chimeric protein rEsat-6 competent cells of Escherichia coli BL21 transform the constructed target plasmid pTSE6. The obtained bacterial strain E. coli BL21/pTSE6 characterized by the following features:

Morphological features. The morphological characteristics of the strain-producer of recombinant protein rEsat-6 is not different from the original strain of Escherichia coli BL21, not containing target plasmid.

Cultural characteristics. On the culture of inim signs producing strains of recombinant protein rEsat-6 is not different from the original strain of Escherichia coli BL21, not containing target plasmid.

Resistance to antibiotics. Cells of strain-producer of recombinant protein rEsat-6 are resistant to ampicillin due to the presence of the target plasmid.

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

The resulting strain deposited at the Institute of culture Collections of microorganisms (research Institute ECR) SRC VB "Vector" under number B-1026.

Chimeric polypeptide rESAT-6 (GST-ESAT-6) (figa and 2B)expressing in the recombinant strain E. coli BL21/pTSE6, contains as a protein carrier N-terminal polypeptide fragment of glutathione-S-transferase unlimited company. (226 S.A. with a molecular mass of 26 kDa) and coupled through the site of hydrolysis by thrombin (LVPI^GS) C-terminal polypeptide fragment of mycobacterial species-specific antigen ESAT-6 (95 S.A. with a molecular mass of 6 kDa), and has the amino acid sequence shown in Figa and 2B.

The invention is illustrated in the following graphics.

Figa. Organization of the recombinant plasmids pTSE6 where: GST - gene protein glutathione-8-transferase; ESAT-6 - cloned fragment, containing the complete gene protein ESAT-6; unique BamHI and EcoRI sites of the restriction endonucleases used in the creation of this innovation is the design; ptac - synthetic promoter present in the vector pGEX-2T and recombinant plasmids; ArRgene β-lactamase (bla gene)providing a transformed bacterial cells resistant to ampicillin.

Figb - restriction map of the target plasmid pTSE6.

Figa and 2B - full nucleotide and amino acid sequence of the recombinant polypeptide GST-ESAT-6 - LVPR^GS amino acid sequence of the site of recognition and hydrolysis by the protease thrombin (highlighted).

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

Example 1. Construction of recombinant plasmids pTSE6.

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

5'-GAGGATCCATGACAGAGCAGCAGTGG-3' [direct primer for exhibiting esxa gene (genes esat6)],

5'-GCGAATTCTAAACACGAGAAAGGGCG-3' [reverse primer exhibiting esxa gene (genes esat6)]

with the temperature-time profile: 94°C - 3 min, 4 cycles [94°C - 30 s, 45°S - 20 s, 72°C - 40], 33 cycle [94°C - 30 s, 58°S - 20 s, 72°C - 40], the resulting annealing 72° - 5 minutes Complete nucleotide sequence of the cloned gene (exhibiting esxa) for the selection of the primary the Oh of the nucleotide sequences of the primers were obtained from the database of the Pasteur Institute (France). The melting temperature of the primers based on the nucleotide sequence was calculated using the NTI program Suite. Annealing of the primers in the first 4 cycles carried out at a low temperature annealing (45° (C)conducting the calculation of the melting temperature of the primers only with a fully hybridization sequences of these primers with genomic nucleotide matrix: 5'-terminal sequence of 8 n is optional and is intended for creation of a site of recognition for the restriction endonucleases BamHI and EcoRI, respectively, to facilitate later cloning.

The reaction mixture comprises polymerase buffer containing 60 mm Tris-HCl, pH 8.5, 25 mm KCl; 1.5 mm MgCl2; 10 mm 2-mercaptoethanol; 0.1% Triton X-100; four deoxynucleosides - dATP, DSTF, dCTP and TTP at the final concentration of each 0.2 mm; primers at a concentration of each of 0.15-0.2 μm and 1 unit of Taq-DNA-polymerase ("Simanim"). The output is about 2 mkg (8 pmol) PCR fragment. The obtained PCR fragment length 388 gel purify presidenial ethanol in the presence of sodium acetate, pH of 4.8, and tRNA as a carrier [14]. The purified preparation of amplicon dissolved in 80 μl of TE buffer and then conducting hydrolysis in EcoRI buffer (SibEnzyme") with the restriction endonucleases BamHI and EcoRI at 37°C for 3-5 h Obtained from the target amplicon rest is ICT with two sticky ends periostat, as described above, is dissolved in 40 ál of TE buffer or bidistilled water and used without additional purification in the reaction ligating split vector molecule (pGEX-2T), also contains two sticky end - BamHI and EcoRI. Mix 2-5 ál of restrict-amplicon (0.5 pmol) with 500 ng (0.15 pmol) of vector DNA pGEX-2T/BamHI/EcoRI in 20 μl ligase buffer (SibEnzyme"), heated at 65°C for 3-5 min, cooled rapidly in ice, add 20 units of DNA ligase of phage T4 and the reaction mixture is transferred in a thermostat, where was incubated over night at 16°C. an Aliquot ligase mixture (1-2 µl) was used to transform competent cells of E. coli strain JM103, cooked the method using a solution of calcium chloride [14], which are sown on plates with agar medium containing ampicillin at a concentration of 50 μg/ml Cup incubated at 37°With during the night. Search for clones containing the desired insert, carried out directly from the colonies using PCR analysis using as amplimers oligonucleotides, complementary to the different circuits of the vector molecule and located on both sides from the cloning of the fragment. While the clones containing the insert full exhibiting esxa gene (genes esat6), after separation of amplificate in agarose gel (1.2 percent) correspond to the length of 548 BP, which exceeds the length of the cloned fragment, as this is t fragment contains a part of the nucleotide sequence of the source vector molecules. After selection of the target plasmids are given in analytical quantities and carry out the necessary restriction analysis (detection of the presence of the restriction sites BamHI and EcoRI). Received target plasmid was designated as pTSE6. Scheme of plasmid DNA presents on Figa and 1B.

Example 2. The receipt of the producer strain on cultivation of which is the production of a chimeric protein GST-genes esat6.

Transformation of cells of the strain E. coli BL21 carried out by the introduction of DNA target plasmids pTSE6 in competent cells prepared by the method using a solution of calcium chloride [14], and then plated on plates with agar medium containing ampicillin at a concentration of 50 μg/ml Cup incubated at 37°With during the night. The appeared colonies are clones of the producer strain.

Cells from each selected clone (usually less than 10) were seeded in 1 ml of liquid medium with ampicillin at a concentration of 50 μg/ml for the night cell cultures and incubated at 37°With no swing. The next day seeded overnight culture of each clone in the ratio of 1:100 2 ml of fresh medium with ampicillin at a concentration of 50 μg/ml and incubated at 37°With swing speed 160 rpm Final selection of clones was carried out by the ability to culture cells to produce recombinant protein rESAT6 in the largest possible number is two after the induction, carried out by adding to a growing culture containing cells of each individual clone and reached a density of 0.15-0.2 at 660 nm, isopropyl-β-D galactopyranoside (IPTG), and further cultivation of the cells at the desired temperature (in this case 37°and 1 mm IPTG). The concentration of the protein produced by cells of the individual clone, evaluate after electrophoresis of lysates of cell cultures in 10%SDS page under denaturing conditions according to laemmli's method.

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

Cells of the strain E. coli BL21 transformed with plasmid pTSE6, grown in LB medium with ampicillin (50 μg/ml) overnight at 37°C. overnight culture (1:100) seeded in 150 ml of LB medium with ampicillin (50 μg/ml) and pokasivaut to early logarithmic phase. Further induce the expression of target protein GST-ESAT-6 by adding IPTG to a concentration of 0.5 mm. Induced cells grow 4 hours at 37°C, then harvested by centrifugation at 5000 g. The expression of the target protein GST-ESAT-6 analyze by electrophoresis according to laemmli's method in 12%polyacrylamide gel and immunoblotting with monoclonal antibodies to protein-media GST. The results of this analysis show the presence of induced culture of E. coli cells BL21/pTSE6 in the processing of cellular precipitation lytic buffer GST-ESAT-6 protein with a molecular mass of about 32 kDa that are appropriate to esthet the calculated molecular mass. In control cell lysates of E. coli BL21 and E. coli BL21/pGEX-2T protein of this molecular weight is missing. This protein is colored in immunoblotting with monoclonal antibodies to protein-media GST.

For analysis, cells resuspended in 8 ml of cold (0° (C) PBS-buffer containing 140 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KN2PO4pH of 7.3, 1 mm protease inhibitor PMSF, and destroy ultrasound in an ultrasonic disintegrator (Ultrasonic processor Cole Farmer) 3×30 c with an interval of 1 min between treatments when the amplitude 50A. To the ultrasound disintegrate add Triton X-100 to 1%, incubated for 30 min at 0°and separating the fraction of soluble proteins from debris by centrifugation at 12000 g for 40 minutes Then mix the supernatant with 200 μl of glutathione-sepharose (Glutathione Sepharose 4B (Amersham, Biosciences AB) and incubated for 30 min with slow stirring at room temperature for affinity binding protein GST-ESAT-6. The mixture is then transferred into microcolony by repeated centrifugation at 600 g, washed several times with column PBS-buffer and perform the elution of the target protein buffer for elution containing 10 mm restored glutathione in 50 mm Tris-HCl, pH 8. The result is a purified preparation of recombinant protein with a concentration of 1 mg/ml

Example 4. Holding IFN-γ analysis.

1st stage (stimulation of the purpose of the th cell culture blood target antigen). Samples of heparinized venous blood of patients is stirred gently in a circular motion for 20 minutes Distribute 100 μl in 96-well plates (Nunc, USA)containing 150 µl/well of complete medium RPMI 1640 with glutamine. Mix thoroughly and make the antigen GST-ESAT-6 in a concentration of 10 μg/ml per well. Separately introduce 100 μl of buffer solution in the wells with zero control (without addition of antigen and mitogen RNA at a concentration of 5 μg/ml per well. Gently mix. Incubated Board 20-48 hours at 37°C in a humid atmosphere containing 5% CO2

2nd stage. To conduct IFN-γ analysis in the control wells tablet with adsorbed monoclonal antibody to gamma interferon production BD Biosciences contribute 100 ál of standard samples of gamma-interferon in different concentrations (from 300-150-75-37,5-18,8-9,4-4,7 PC/ml) - production of BD Biosciences. In the remaining wells tablet make 100 μl samples of blood plasma after stimulation, placed in a shaker and incubated for 2 h at room temperature. After incubation the unbound washed 3 times with wash buffer (FSB-T). After washing each well of the tablet make 100 ál of conjugate biotinylated antibodies to IFN-γ production BD Biosciences and incubated for 60 min at room temperature. At the end of incubation, washed tablet 3 times b the atmospheric solution FSB So Then to each well of the tablet add 100 ál of conjugate avidin-horseradish peroxidase production BD Biosciences working dilution. Incubate 60 min at room temperature. After incubation the unbound washed 5 times with wash buffer (FSB-T) and 3 times with N2About distilled. Then bring in each well of the tablet 100 μl of a solution of TMB (3,3',5,5'-tetramethylbenzidine), put the tablet in the dark place, and incubated at room temperature for 30 minutes the Reaction terminorum added to each well of the tablet in 50 µl of stop solution (0,9 N solution of H2SO4). Measure the optical density on automatic spectrophotometer type MultiScan" at a wavelength of 450 nm.

To assess the concentration of IFN-γ in the samples using calibration curve in logarithmic coordinates: the x - axis the concentration of IFN-γ (PCG/ml), the y-axis is the value of optical density of the sample. According to the obtained values are conducting a calibration curve and calculate the quantitative content of IFN-γ in PG/ml or IU/ml in the test and control samples. The results are calculated from the mean OD in three holes (ρ<0,05). The Cutoff values for IFN-γ>300 PG/ml is considered positive.

Example 5. Mononuclear cells whole cell cultures of blood (2×105cells/well)isolated potangaroa procedure using gradient centrifugation in the presence of Ficoll-Hipac 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 complete medium RPMI 1640 production of SRC VB "Vector". Then to each well of the tablet make chimeric antigen GST-ESAT-6 in a concentration of 5-10 µg/ml; in the control wells tablet make mitogen (Con a or RNA) in a concentration of 4 μg/ml in triplicate. The final volume of the reaction medium in the well of the tablet is 250 µl. Incubated Board 16-24 hours at 37°C in a humid atmosphere containing 5% CO2.

To conduct IFN-γ analysis in the control wells tablet with adsorbed monoclonal antibody to gamma interferon make 100 ál of standard and control samples. In the remaining wells tablet make 100 μl samples of blood plasma after stimulation. Put in a shaker and incubated for 2 h at room temperature. After incubation the unbound washed 3 times with wash buffer (FSB-T). After washing each well of the tablet make 100 ál of conjugate biotinylated antibodies to IFN-γ and incubated for 60 min at room temperature. At the end of incubation, washed tablet 3 times with the buffer solution of the FSB, then to each well of the tablet dobavlaut 100 ál of conjugate avidin-horseradish peroxidase in the working dilution and incubated for 60 min at ControlTemplate. After incubation the unbound washed 5 times with wash buffer (FSB-T) and 3 times with distilled water. Then to each well of the tablet make 100 μl of a solution of TMB (3,3',5,5'- tetramethylbenzidine), put the tablet in the dark place, and incubated at room temperature for 30 minutes the Reaction terminorum added to each well of the tablet in 50 µl of stop solution (0,9 N solution of H2SO4). The optical density of samples was measured on an automated spectrophotometer type MultiScan" at a wavelength of 450 nm.

To assess the concentration of IFN-γ in the samples using calibration curve in logarithmic coordinates: the x - axis the concentration of IFN-γ (PCG/ml), the y-axis is the value of optical density of the sample. According to the obtained values are conducting a calibration curve and calculate the quantitative content of IFN-γ in PG/ml or IU/ml in the test and control samples. The results are calculated from the mean OD in three holes (ρ<0,05). The Cutoff values for IFN-γ>300 PG/ml is considered positive.

Example 6. Sorption µa to IFN-γ production BD OptEIA Reagent carried out using 0.1 M carbonate buffer, pH 9.5 V for 16 h at 4°on the plates (Nunc, MaxiSorb, USA). After flashing the tablet wash buffer (FSB, pH 7.0) 3 times with 300 µl/well hold the lock behavior of the displacement of the hole blocking buffer solution FSB, pH 7.0, containing 10% fetal serum for 60 min at room temperature. Followed by washing of the tablet, as described above.

The prepared tablet with sorbed µa to IFN-γ use when conducting IFN-γ-analysis. For this purpose, in each well of the tablet bring in 150 μl of complete medium RPMI 1640. Samples of heparinized venous blood of patients gently mixed and then distributed to 100 μl in 96-well plates (Nunc, USA). Mix thoroughly and make a solution of antigen GST-ESAT-6 in RPMI 1640 with 0.2 mm glutamine at a concentration of 10 μg/ml per well. In wells a-1, B-1 and C-1 contribute mitogen (RNA), in wells D-1, E-1 add 100 ál of diluent buffer solution (zero control). Gently mix. Incubated Board 24-48 hours at 37°C in a humid atmosphere containing 5% CO2when mixing. After incubation the unbound washed with 5×300 μl/well of distilled water and 5×300 ál FSB-T buffer solution. To construct the calibration graph when conducting IFN-γ analysis previously conducted incubation with standard calibration samples IFN-γ. To do this in the control wells tablet with adsorbed monoclonal antibody to gamma interferon production BD Biosciences contribute 100 ál of standard samples of gamma-interferon in different concentrations (from 300-150-75-37,-18,8-9,4-4,7 PC/ml) - production BD Biosciences or 100 ál of standard calibration samples containing IFN-γ (2000-1000-500-300-100-50-0 PCG/ml) and control sample (330 PCs/ml IFN-γ) manufactured by "Vector-best". Incubated according to the instructions for use of the kits. After incubation the strips tablet washed 3 times with wash buffer (FSB-T). After washing each well of the tablet make 100 ál of conjugate biotinylated antibodies to IFN-γ and incubated for 60 min at room temperature. After incubation the unbound washed 3 times with buffer solution FSB-T, to each well of the tablet add 100 ál of conjugate avidin-peroxidase or streptavidin-horseradish peroxidase in the working dilution and incubated for 60 min at room temperature. After incubation the unbound washed 5 times with wash buffer (FSB-T) and 3 times with distilled water and applied to each well of the tablet 100 μl of a solution of TMB (3,3',5,5'- tetramethylbenzidine). The tablet is placed in a place protected from light and incubated at room temperature for 30 minutes the Reaction terminorum added to each well of the tablet in 50 µl of stop solution (0,9N solution of H2SO4) and measure the optical density on automatic spectrophotometer type MultiScan" at a wavelength of 450 nm. The results of the analysis represent the Cutoff values that receive the in-built calibration curves as described above.

Tuberculosis control is carried out to detect active TB and screening of persons having contact with the use of tuberculin tests. Fast way early detection of infection in the blood using recombinant proteins is an alternative skin test. Identifying the level of IFN-γ correlates with the stage and extent of TB infection, the level of immune response and the probability of progression to active TB. Table 1 presents the results of detection of active tuberculosis with the use of IFN-γ-analysis, as well as among healthy population. The conducted studies show that received recombinant proteins help to identify active tuberculous process among patients with pulmonary tuberculosis. The level of IFN-γ among healthy contingent does not exceed limit values (>300 PG/ml IFN-γ)corresponding risk or TB patients. These data demonstrate the specificity of the method. Clinical status of patients with different forms of tuberculosis infection identified using hybrid proteins rESAT-6, presented in Table 3. Table 4 presents the results of a comparative study of the specificity and sensitivity of recombinant proteins rESAT-6 compared to PPD-tuberculin stimulation of whole cell cultures of blood. pacificnet recombinant proteins in IFN-γ analysis of TB is higher than the PPD tuberculin.

Table 1

IFN-γ - analysis of tuberculosis during stimulation of the hybrid proteins in patients with active form of tuberculosis
Patients with active TBIFN-γ (PG/ml)
RESAT-6ConARNAPPD
11750670047824100
22360732454213650
3110453032182850
44475730067104870
51320965089714640
627786187646200
74210123441423017320
8760485143523980
9324019870213405830
103420183001723510350
112290165401453021200
12131812347132475730
13642345032410670
145640176501893015340
151764098523
166423410287507458
173340193401874510480
18168034500295606130
Donors<300<300<300180-632

The Cutoff values for IFN-γ>300 PG/ml - positive. The results were calculated from the mean OD in three holes (ρ<0.05) and expressed in PG/ml using the calibration graph.

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

57,4
Table 2.

IFN-γ - analysis of pulmonary tuberculosis during stimulation of the hybrid proteins and mitogen -
Patients with active

TB
IFN-γ (IU/ml)
PPDrESAT-6Mitogen
RNA
TB54,32,3463,5
TB27,03,147,6
TB59,2the 5.7123,0
TB73,03,2117,0
TB24,82,743,2
TB11,71,7323,1
TBthe 4.70,87to 126.8
TB2,90,8121,3
TB5,30,7353,1
TB27,60,83143,9
TB57,20,2365,3
TB10,96,472,1
TB62,37,2
TB1,72,119,7
TBthe 33.41,734,5
TB7,213.267,3
TB98,712,478,1
TB74,619,332,3
TBto 43.1132,2179,3
TB67,3147,5165,7

Table 3.

The clinical status of TB cases detected using recombinant proteins rESAT-6 and IFN-γ analysis
Order to show s/pTB infectionSensitivity (%)Specificity (%)
1Infiltrative tuberculosis of the lungs86-9587-97
2Focal pulmonary TB87-9493-94
3Disseminated tuberculosis84-97 86-94
4Fibrous-cavernous lung tuberculosis96-10097-100
Table 4.

The sensitivity and specificity of the PPD, rESAT-6 in TB patients and healthy donors
The number of IFN-γ PCG/mlAntigenThe number of identified TB/to the total number.Sensitivity (%)*Specificity (%)**
TBHealthy ***
>300PPD5/67/7830
rESA Vol-64/64/147371
>1000rESA9/103/219085,7
T-6 PPD7/913/1478,77
>3000rESA Vol-6 17/212/1980,9to 89.5
* Positive response among TB-infected patients

** Positive response in uninfected patients.

*** - BCG-vaccinated and unvaccinated patients

Some forms of TB (acute progressive) cannot be detected by the method of screening. Patients with such processes fall mainly in the General hospital, where the diagnosis is sometimes difficult because of the similarity of the x-ray pictures with nonspecific lung diseases. The only reliable method of diagnosis in such cases is a direct sputum smear microscopy or use of IFN-γ-analysis. All investigated groups of patients with pulmonary TB had a positive reaction using microscopic methods, culture, x-ray and molecular biology (PCR analysis).

LITERATURE

1. Nuket D., Stephen L.J. // Development of a human gamma interferon enzyme immunoassay and comparison with tuberculin skin testing for detection of Mycobacterium tuberculosis.// Infection Clinical and Diagnostic Laboratory Immunology, 1998, V.5, No. 4, R-536.

2. Nakamura R., Velmonte M.A., KawajiriK, Ang C.F., Frias R.A., Mendoza M.T. et al. // MPB64 mycobacterial antigen: a new skin-test reagent through patch method for rapid diagnosis of active tuberculosis. // Int J Tuberc Lung Dis, 1998, №2 (7), R-546.

3. Wilkms EGL. // Antibody detection in tuberculosis. // In: Daves PDO, ed. Clinical tuberculosis, 2nded. London: Chapman and Hall, 1998, p.81-95.

4. Imaz M.S., Zerbini E. // Antibody response to culture filtrate antigens of Mycobacterium tuberculosis during and after treatment of tuberculosis patients. // Int J Tuberc Lung Dis., 2000, №4(6), p.562-569.

5. Sonnenberg M.G. and J.T. Belisle // Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry. // Infect. Immun., 1997, V 65, No. 11, p.4515-4524.

6. Smith S.M., R. Brookes, M.R. Klein et al. // Human CD8+ CTL specific for themycobacterial major secreted antigen 85 A. // J. Immunol., 2000, No. 165, p.7088-7095.

7. Reinout van Crevel, Tom H.M. Ottenhoff and Jos W.M. van der Meer // Innate Immunity to Mycobacterium tuberculosis. // Clinical Microbiology Reviews, 2002, V.15, n 2, R-309.

8. Chackerian A.A., Perera T.V. et al. // Gamma Interferon-Producing CD4+ T Lymphocytes in the Lung Correlate with Resistance to Infection with Mecobacterium tuberculosis. // Infection and Immunity, 2001, V.69, no .4, p.2666-2674.

9. Ansar A.P., K.A. Wilkinson, Klenerman P., McShane H., R.N. Davidson, Pasvol g, Hill, A.V.S. and Lalvani A. // Direct Ex Vivo Analysis of Antigen-Specific IFN-γ-Secreting CD4 T Cells in Mycobacterium tuberculosis-Infected Individuals: Associations with Clinical Disease State and Effect of Treatment. // The J. of Immunol., 2001, V.167, p.5217-5225.

10. Sodhi A., J. Gong, C. Silva, D. Qian, Bames P.P. // Clinical correlates of interferon gamma production in patients with tuberculosis. // Clin Infect Dis., 1997, №25 (3), p.617-620.

11. Sorensen A.L., S. Nagai, Houen G., Andersen p, Andersen A.B. // Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. // Infect Immun, 1995, No. 63, p.1710-1717.

12. Harboe, M., Oettinger So, H.G. Wiker, Rosenkrands I, Andersen P. // Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG. // Infect Immun 1996, No. 64, p.16-22.

13. Harboe, M., G. Wiker, Ulvund A.S., H. Malin, Dockrell A.., Holm M.C., P. Andersen // B-cell epitopes and quantificaton of the ESAT-6 protein of Mycobacterium tuberculosis. II Infect Immun, 1998, No. 66, p.717-723.

14. Maniatis T., Fritsch E., Sambrook J. Methods of genetic engineering. Molecular cloning. M.: Mir. 1984.

1. Recombinant plasmid DNA pTSE 6, encoding a hybrid polypeptide GST-ESAT-6 with the properties of species-specific mycobacterial antigen ESAT-6, with a molecular weight of 3.45 MDA has a size 5316 P.N. and consists of the following elements:

EcoRI-BamHI fragment of the vector plasmid pGEX-2T (Pharmacia Biotech) size 4938 gel containing gene β-lactamase induced by the TAC promoter, the internal lacIq- the gene encoding the protein is a repressor of Lac operon, a gene fragment glutathione-S-transferase from Schistosoma japonicum (GST) with multiple site for cloning genes in the 3'end of this gene and the site of proteolysis by thrombin in the same reading frame;

EcoRI - BamHI fragment size 378 gel containing flanked by the restriction sites EcoRI and BamHI full gene esat-6, obtained by amplification of a fragment of the genome of Mycobacterium tuberculosis;

contains:

as a genetic marker gene β-lactamase determining the stability of the transformed plasmid pTSE 6 E. coli cells 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 N And CMC SRC VB "Vector" under number B-1026, producer of 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 of Mycobacterium tuberculosis ESAT-6, which can be used for early species-specific diagnosis of tuberculosis infection in IFN - analysis of expressed recombinant Escherichia coli BL21/pTSE6 according to claim 2, contains as a protein carrier N-terminal polypeptide fragment of glutathione-S-transferase unlimited company. (232 S.A. with a molecular mass of 26 kDa) and coupled through the site of proteolysis by thrombin (LVPR^GS) C-terminal polypeptide fragment of mycobacterial species-specific antigen ESAT-6 (95 S.A. with a molecular mass of 6 kDa), and has the amino acid sequence shown in figure 2.



 

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