RU2381274C2 - RECOMBINANT PLASMID DNA pTB232, WHICH CODES HYBRID POLYPEPTIDE GST-CFP10 WITH PROPERTIES OF SPECIES-SPECIFIC MYCOBACTERIAL ANTIGEN CFP10, RECOMBINANT STRAIN OF BACTERIA Escherichia coli-PRODUCER OF HYBRID POLYPEPTIDE GST-CFP10 AND RECOMBINANT POLYPEPTIDE GST-CFP10 - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention is related to production of new hybrid polypeptide GST-CFP10 by microbiological synthesis with properties of species-specific protein-antigen CFP10 Mycobacterium tuberculosis, which may be used for early species-specific diagnostics of tuberculosis infection. Recombinant plasmid DNA pTB232 has been constructed, which codes hybrid polypeptide GST-CFP10 with properties of mycobacterial antigen CFP10, with average molecular weight (m.w.) 3.4 MDa and having size of 5257 p.n. Recombinant strain of bacteria E. coli BL21/pTB232 contains recombinant plasmid DNA pTB232, is producer of hybrid polypeptide GST-CFP 10 with properties of mycobacterial antigen CFP10 and is deposited in KM GNC VB "Vector" under number B-1027. Recombinant polypeptide GST-CFP10, produced with strain of bacteria E. coli BL21/pTB232, contains as protein-carrier N-end polypeptide fragment glutathione S-transferase S.j. (226 a.o. with m.w. of 26.3 kDa), joined via end site of thrombin hydrolysis (LVPR^∧GS) with C-end polypeptide fragment of antigen CFP10 (100 a.o. with m.w. of 10.8 kDa) and has complete aminoacid sequence with length of 326 a.o. and m.w. of 37.1 kDa, given in text of description.

EFFECT: use of invention provides for the possibility to produce target highly pure hybrid polypeptide GST-CFP10 in preparative amounts with preservation of immunogenic properties of the latter.

3 cl, 4 dwg, 4 tbl, 6 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-CFP10 polypeptide with the properties of the species-specific antigen protein Mycobacterium tuberculosis CFP10, 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 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 obtaining false-positive results in the Mantoux reaction is associated with the manifestation of cross-reactivity to non-tuberculous mycobacteria. About 10% of individuals are reactive to non-tuberculous mycobacteria, of which 30% are positive in a skin test.

Among the classical methods for the diagnosis of tuberculosis, the method of identifying the pathogen culture 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 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 of the procedures used, high sensitivity and specificity among infected individuals [3]. Serological methods are very diverse. The sensitivity and specificity of tests using enzyme-linked immunosorbent assay (ELISA) technology varies over a fairly wide range and is 40-85% and 67-100%, respectively. On the one hand, this is due to the fact that the indicators of the humoral immune response in tuberculosis are often reduced, and this, in turn, leads to the fact that antibodies obtained in the human body to 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. It is the latter factor that is the main factor in identifying markers of humoral immunity in patients with tuberculosis, since a decrease in the level of antibodies entails a decrease in the proportion of positive results in ELISA, which 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 has similarities with proteins of other species of the genus Mycobacterium and even bacteria of other genera. A certain amount of culture filtrate proteins secreted by M. tuberculosis mycobacteria and possessing immunogenic and enzymatic activity are 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 (eg, CD4, CD8) [6]. For example, CB4-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 stimulation of T-cell effectors in the blood, 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 reveal a lower percentage of vaccinees who have a positive result for the presence of tuberculosis infection than the skin test data. 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 differentiating 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 active forms of tuberculosis.

The known protein CFP10, first identified by Berthet FX et al. [11], is highly specific for virulent species of tuberculosis pathogens. As a native protein, CFP10 was found in the cytosolic fraction and the cell wall of mycobacteria, as well as in the culture fluid. The molecular weight of the purified native protein is 14 kDa. The main advantage of this protein is that there is no nucleotide sequence encoding it in the genomes of vaccine BCG strains [12]. CFP10 is the main antigen causing the development of an early T-cell response. This process is accompanied by increased production of γ-IFN. The CFP10 protein gene is part of the 9.5 kbp RD1 genomic fragment present in the genomes of M. tuberculosis and M. bovis strains, including laboratory strains of M. tuberculosis (H37Rv, H37Ra). But this nucleotide sequence (RD1 region) is absent in all vaccine strains of M. bovis BCG, which means that CFP10 is also absent in vaccine preparations, which allows its use for diagnostic purposes [12].

Isolation and purification of CFP10 protein from mycobacterial culture filtrate is an expensive and complex procedure, while the product yield is extremely low. That is why the use of recombinant DNA technology to obtain the specified protein for practical diagnostic purposes is economically more appropriate.

Known genetic engineering method of producing a recombinant polypeptide with the properties of the mycobacterial antigen CFP10 used for diagnostic purposes [11]. In this case, the target protein is obtained with 6 histidine (6 His) amino acid residues at the N-terminus.

The closest analogue (prototype) is the invention according to the patent of the Russian Federation No. 2277540, in which the objects of patenting are:

- a hybrid protein containing the complete protein CFP10 from M. tuberculosis, fused to the complete protein ESAT6 from M. tuberculosis through a linker amino acid sequence;

- recombinant plasmid DNA pTBD16 for expression of the hybrid protein CFP10-ESAT6.

- E. coli strain DLT1270, transformed with the obtained recombinant plasmid DNA pTBD16 - producer of the hybrid protein CFP10-ESAT6 [14].

However, the proposed design does not allow to produce preparative amounts of this polypeptide. In addition, during biotechnological purification of the obtained recombinant CFP10 polypeptide, the latter undergoes significant degradation, which affects its immunogenic properties.

The technical result of the invention is the creation of a recombinant strain of bacteria Escherichia coli, a producer of a hybrid polypeptide with the properties of the species-specific mycobacterial antigen CFP10, which has such a design that would allow to produce the target highly purified polypeptide in preparative quantities while maintaining the immunogenic properties of the latter.

This result is achieved by constructing a recombinant plasmid DNA rTB232 encoding a chimeric polypeptide: glutathione-S-transferase (GST) + CFP10 (hereinafter rCFP10). In this case, the gene encoding the polypeptide with the properties of the mycobacterial antigen CFP10 is in the same reading frame with the glutathione S-transferase gene from Schistosoma japonicum (GST S.j.) (Fig. 1A and B).

Recombinant plasmid DNA rTB232 (Figa and 1B) with an average molecular weight of 3.4 MDa has a size of 5257 bp (images of plasmid construction, restriction map, size calculation were performed using the NEBcutter V2.0 (UK) program in on-line mode (http://tools.neb.com/NEBcutter2/index.php)) and consists of the following elements:

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

- 319 bp EcoRI-BamHI fragment containing the complete CFP10 protein gene flanked by restriction endonuclease sites EcoRI and BamHI, obtained by amplification of the corresponding gene fragment with M. tuberculosis genomic DNA;

contains:

- as a genetic marker, the β-lactamase gene, which determines the resistance of E. coli cells transformed with plasmid rTB232 to the antibiotic ampicillin;

- unique restriction sites: BamHI - 930/934 , EcoRI - 1249/1253 (the positions of cleavage by the corresponding restriction endonucleases on both chains are indicated, the numbering is carried out on one chain).

The presence of a GST fragment in the recombinant rCFP10 polypeptide allows the preparation of preparative amounts of the target protein using affinity chromatography. These structural properties are important for the biotechnological cycle associated in the selection of recombinant protein. It is the presence of the GST fragment that facilitates the purification of the recombinant polypeptide and obtain a highly purified protein with the properties of the mycobacterial CFP10 antigen. In the form of a recombinant fusion protein, rCFP10 does not undergo degradation and is significantly more stable than the analogous one that we obtained earlier in the C-terminal part of ESAT-6 (RF patent No. 2282661). Apparently, the presence of the GST fragment in the composition of the chimeric protein allows it to be protected from the action of blood enzymes during stimulation of a whole cell blood culture in the process of diagnostic analysis. The prolongation of its action upon stimulation of whole blood or mononuclear cells of whole blood using such structures is an important factor for the stability of 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 CFP10 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 CFP10 protein and lacking the functional activity of the GST fragment. Located between the GST polypeptide fragments and CFP10 (Figure 2), the thrombin hydrolysis site allows, if necessary, to obtain the mycobacterial CFP10 antigen free of the N-terminal GST polypeptide fragment. The recombinant CFP10 antigen at the N-terminus after thrombin cleavage will contain only two additional amino acid residues: glycine is the N-terminal, serine is the second amino acid residue, followed by the amino acid sequence that strictly corresponds to the target protein CFP10.

To obtain a bacterial strain producer of the chimeric rCFP10 protein, competent E. coli BL21 cells are transformed with the constructed target plasmid rTV232. The resulting bacterial strain E. coli BL21 / pTB232 is characterized by the following features:

Morphological signs. According to morphological characteristics, the producer strain of the recombinant protein rCFP10 does not differ from the original E. coli strain BL21 that does not contain the target plasmid.

Cultural signs. According to cultural characteristics, the producer strain of the recombinant protein rCFP10 does not differ from the original E. coli BL21 strain that does not contain the target plasmid.

Antibiotic resistance. The cells of the producer strain of the recombinant rCFP10 protein exhibit ampicillin resistance due to the presence of the target plasmid.

A significant difference between the E. coli strain BL21 / pTB232 is that it provides the synthesis of the rCFP10 chimeric polypeptide (GST-CFP10) with the properties of the mycobacterial CFP10 antigen with an expression level of 1.8-12 mg / ml protein.

The resulting strain was deposited in the Collection of microorganism cultures (CCM) of the Federal State Institution Scientific Center of the WB "Vector" of Rospotrebnadzor under the number B-1027.

The chimeric rCFP10 polypeptide (GST-CFP10) (Figure 2) produced by the recombinant E. coli BL21 / pTB232 strain contains an N-terminal polypeptide fragment of glutathione S-transferase S.j. as a carrier protein. (226 a.s. with a molecular weight of 26.3 kDa; calculation was performed using the Compute pl / Mw tool (Switzerland) software on-line (http://cn.expasy.org/tools/pi tool.html) connected through its terminal site of thrombin hydrolysis (LVPR ^ GS) to the C-terminal polypeptide fragment of the species-specific mycobacterial antigen CFP10 (100 a.a. with a molecular weight of 10.8 kDa; the calculation was performed using the Compute pl / Mw tool), and has a complete amino acid sequence (326 aa, 37.1 kDa; calculation was performed using the Compute pl / Mw tool program) shown in FIG. 3.

The invention is illustrated by the following graphic materials.

Figa. Scheme of the organization of the recombinant plasmid rTV232, where GST is the gene for the protein glutathione-3-transferase; CFP10 — cloned fragment obtained by PCR amplification and including the full gene for the CFP10 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 transformed bacterial cells with resistance to ampicillin.

Figb. Restriction map of the target plasmid rTV232. Image of the construction of plasmid rTV232 (5257 bp). b - fusion recombinant protein GST-CFP10 size 326 A.O. Images were constructed using the NEBcutter V2.0 (UK) software (http://tools.neb.com/NEBcutter2/index.php) [Vincze, T., Posfai, J. and Roberts, R.J. NEBcutter: a program to cleave DNA with restriction enzymes. Nucleic Acids Res. 31: 3688-3691 (2003)], available online.

Figure 2. Docking sites during the insertion of the esxB gene of plasmid pTB232 into the glutathione S-transferase (gst) gene as compared to the same site in the initial (vector) plasmid pGEX-2T. NP is the nucleotide sequence, AP is the amino acid sequence. The first codon of the insertion protein is shown after the nucleotide sequence of the thrombin proteolysis site, the stop codon is underlined at the end of the insertion gene.

- аминокислотная последовательность сайта узнавания и гидролиза протеазы тромбин (выделен цветом).Figa and B. The complete nucleotide and amino acid sequence of the recombinant GST-CFP10 polypeptide, where

- the amino acid sequence of the site of recognition and hydrolysis of the thrombin protease (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 rTB232

The plasmid pGEX-2T (Pharmacia) and strains: JM103 E. coli {Δ (lac-proAB), thi, strA, supE, endA, sbcB, hsdR ^- , [F 'traD36, proAB, lacI ^q , ZΔM15]}; BL21 E. coli B {F ^- , dcm, ompT, hsdS (r _B - m _B -), gal}.

The genomic M. tuberculosis H37Rv DNA isolated from inactivated mycobacteria cells is used as a source of nucleotide material (matrix) to obtain a cloned amplifiable fragment using polymerase chain reaction (PCR). The gene encoding the CFP10 protein is amplified using primers:

5'-GA GGATCC ATGGCAGAGATGAAGACC-3 '(direct primer for the esxB gene); 5'-GC GAATTC TATTAGCGGGTCAGAAGC-3 '(reverse primer for the esxS gene). PCR is carried out in a volume of 50 μl on a Tertsik thermocycler (DNA-technology, Moscow) with a temperature-time profile: 94 ° C for 3 min; 5 cycles [94 ° C - 30 s, 45 ° C - 20 s, 72 ° C - 40 s], 30 cycles [94 ° C - 30 s, 58 ° C - 20 s, 72 ° C - 40 s], 72 ° C - 3 min. The complete nucleotide sequence of the cloned gene (esxB) for selection of the primary nucleotide sequence of the primers was extracted from the database (DB) of the Pasteur Institute (http://genolist.pasteur.fr/TubercuList/) (France). The melting points of the primers are selected based on the extended nucleotide sequence obtained from the database, including the full esxB gene, using the NTI Suite 8. The primers are annealed in the first 5 cycles at a low annealing temperature (45 ° C), since the 5'-terminal nucleotide sequence 8 n.d., in the structure of which a restriction site is laid (in this case, BamHI or EcoRI), cannot form a perfect hybrid duplex with the DNA matrix (M. tuberculosis) at the initial instant of PCR after a longer 3'- end part primer formed it. In this case, the calculation of the melting points of each primer is carried out only with a fully hybridizing sequence, discarding the first 8 n.a. from the structure of the primer. The melting points for the primers were calculated using the NTI Suite 8 program or the Oligonucleotide Properties Calculator (http://www.basic.northwestern.edu/biotools/oligocalc.html) (USA). 5'-terminal sequence 8 N.O. is complementary to the main nucleotide sequence of each primer and is intended to create a recognition site for BamHI and EcoRI restriction endonucleases, respectively, in order to facilitate the subsequent cloning step.

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; 4 deoxynucleoside triphosphates - dATP, dGTF, dTTP and TTF - with a final concentration of each 0.2 mM; primers with a concentration of each 0.15-0.25 μm and 5 units Taq DNA polymerase (Sibenzyme, Novosibirsk, Russia). The yield is about 2 μg (8 pmol) of the PCR fragment. The resulting PCR fragment (amplicon) 329 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 is 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 fragment obtained from amplicon with two sticky ends reprecipitated as described above, dissolved in 40 μl of TE buffer or bidistilled water and used without further purification in the ligation reaction with the digested vector molecule (pGEX-2T) using restriction endonucleases BamHI and EcoRI, 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 plated 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 as amplifiers complementary to different chains of the vector molecule and located on both sides of the fragment cloning site. In this case, clones containing the embedding of the complete esxB gene (cfp10), after separation of the amplification by agarose gel electrophoresis (1.2%) in the presence of ethidium bromide, show a band stained under ultraviolet light corresponding to a length of 489 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 rTB232. Scheme of plasmid DNA is presented in Figa and 1B.

Example 2. Obtaining a producer strain, the cultivation of cells which produces chimeric protein GST-CFP10

Cell transformation of E. coli strain BL21 is carried out by introducing the DNA of the target plasmid pTB232 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 50 μg / ml ampicillin. 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 YT 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 YT medium with ampicillin at a concentration of 50 μg / ml and incubated at 37 ° C with a swing speed of 160 rpm to a density of D ₅₅₀ = 0.5-0 ,8. The expression of the esxB gene is induced by the addition of isopropyl-β-D-galactopyranoside (IPTG) to a final concentration of 0.5-1.0 mM, incubated at 37 ° C with vigorous aeration (160-170 rpm) for 3-5 hours then aliquots of cell suspensions of each clone are selected and the lysates obtained from cell cultures are analyzed by electrophoresis in 10% SDS page under denaturing Laemmli conditions. The final selection of clones is carried out according to the ability of the cell culture of each individual clone to produce the maximum possible amounts (after induction) of the recombinant protein rCFP10, the amount and concentration of which is estimated by the electrophoretic picture (in our case, this is 37 ° C and 1.0 mM IPTG).

Example 3. The selection of recombinant protein GST-CFP10

Cells of E. coli strain BL21 transformed with plasmid pTB232 are 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 a density of D ₅₅₀ = 0.8 for 3 hours. Next, expression of the target GST-CFP10 protein is induced by adding IPTG to a concentration of 1, 0 mM. The induced cells grow at 37 ° C for 5 h, after which the cell culture is cooled to 0 ° C, and the cells are harvested by centrifugation at 5000 g for 15 min at 4 ° C. The expression of the target GST-CFP10 protein was analyzed by Lammley electrophoresis on a 12% polyacrylamide gel and immunoblot with monoclonal antibodies to the GST carrier protein. The results of this analysis show the presence of strain BL21 / pTB232 in the E. coli cell culture induced when processing cell sediments with GST-CFP10 lysis buffer protein with a molecular weight of about 37 kDa, which corresponds to the calculated molecular weight. In control lysates of E. coli BL21 cells and E. coli BL21 / pGEX-2T, there is no protein with this molecular weight (according to electrophoresis). This protein is stained by immunoblotting with monoclonal antibodies to the GST carrier protein.

For further analyzes, 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 is destroyed by ultrasound on an ultrasonic disintegrator (Ultrasonic processor Cole Parmer) 3 × 30 s with an interval of 1 min between treatments at an amplitude of 50 A. To the ultrasonic disintegrate add Triton X-100 to 1%, incubate for 30 minutes at 0 ° C and the soluble protein fraction is separated from the debris by centrifugation at 12000g for 40 minutes 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-CFP10 protein. The mixture was then transferred to a microcolumn by repeated centrifugation at 600 g, the column was washed several times with PBS- and PBS buffer, and the recombinant target protein was eluted with elution buffer containing 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0. The result is a purified recombinant protein preparation with a concentration of 1.5 mg / ml.

Example 4. The IFN-γ analysis

Stage 1 (stimulation of a whole cell culture of blood with the target antigen) Samples of the 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 RPMI 1640 medium with L-glutamine. Mix well and add GST-CFP10 antigen 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. Incubate tablets 24-48 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 various concentrations (from 300-150-75-37.5-18.8-) are introduced into the control wells of a plate with sorbed monoclonal antibodies to gamma interferon manufactured by BD Biosciences. 9.4-4.7 pc / ml). 100 μl of the whole blood samples to be studied are added to the remaining wells after stimulation, placed in a shaker and incubated for 2 hours at room temperature. After incubation, the wells are washed 3 times with wash buffer (PBS-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 plates are washed 3 times with FSB-T buffer solution, after which 100 μl of horseradish avidin peroxidase conjugate manufactured by BD Biosciences in working dilution are added to each well of the tablet. Incubated for 60 minutes at room temperature. After incubation, the plates are 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 plates are 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 concentration of IFN-γ (pg / 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. Results are calculated from the average OD in three wells (p <0.05). Cutoff values for IFN-γ> 300 pg are considered positive.

Example 5. Mononuclear cells of whole cell blood culture (2 × 10 ⁵ cells / well), isolated according to the standard procedure 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 complete medium RPMI 1640 produced by the State Research Center of the World Bank "Vector". Then, chimeric antigen GST-CFP10 at a concentration of 5-10 μg / ml is added to each well of the plate; 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 were 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 were added to the control wells with 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 plates are 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 plates are washed 3 times with FSB-T buffer solution, after which 100 μl of horseradish avidin peroxidase conjugate is added to each well of the plate in working dilution and incubated for 60 min at room temperature. After incubation, the plates are 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 tablets are 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. Results are calculated from the average OD in three wells (p <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 9.6 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 blocking buffer solution FSB, pH 7.0, containing 10% fetal serum, for 60 minutes at room temperature, after which wash the tablet, 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 add a solution of GST-CFP10 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. Gently mix, incubate the plates for 24-48 hours at 37 ° C in a humid atmosphere containing 5% CO ₂ with stirring. After incubation, the plates are 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 pg / ml) -production of BD Biosciences or 100 μl of standard calibration samples containing IFN-γ (2000-1000-500-300-100-50-0 pg / ml) and a control sample (330 pg / 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 plates are 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 plates are washed 5 times with wash buffer (FSB-T), 3 times with distilled water, and then 100 μl of TMB solution (3.3 ', 5.5'-tetramethylbenzidine) is added to each well of the plate. The tablets are placed in a dark place and kept at room temperature for 30 minutes. The reaction is terminated by adding to each well of the plate 50 μl of a stop reagent (0.9 N solution of H ₂ SO ₄ ) 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 a healthy contingent. As the studies show, the resulting recombinant protein allows you 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 the rCFP10 fusion protein is presented in Table 3. Table 4 presents the results of a comparative study of the specificity and sensitivity of the recombinant rCFP10 protein compared to PPD-tuberculin during stimulation of whole cell blood culture. The specificity of the recombinant protein in the IFN-γ analysis of tuberculosis is higher than the PPD-tuberculin.

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 subjects (the results are presented in table 2).

Some forms of tuberculosis (acutely progressive) cannot be detected during preventive examinations. Patients with such processes fall mainly in general hospitals, where their diagnosis is often 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).

Table 1 IFN-γ analysis of tuberculosis with stimulation of PPD, rCFP10 fusion protein and mitogens in patients with active pulmonary tuberculosis The patients IFN-γ (pg / ml) PPD rCFP10 Mitogen Cona PHA 21 4100 845 6700 4782 22 3650 1985 7324 5421 23 2850 55 4530 3218 24 4870 3200 7300 6710 25 5110 2755 9650 8971 26 6200 2849 7861 8764 27 17320 5780 12344 14230 28 3980 67 4851 4352 29th 5830 4210 19870 21340 thirty 10350 7400 18300 17235 31 21200 415 16540 14530 32 5730 2065 12347 13247 33 670 57 23450 32410 34 15340 3875 17650 18930 35 23 19 640 985 36 7458 2190 23410 28750 37 10480 6240 19340 18745 38 6130 1745 34500 29560

Cutoff values for IFN-γ> 300 pg / ml were considered positive, the results were calculated from the average of three wells (ρ≤0.01) and expressed in pg / ml using a calibration graph.

table 2 IFN-γ analysis of pulmonary tuberculosis stimulated by PPD, rCFP10 fusion protein and mitogens The patients IFN-γ (IU / ml) PPD rCFP10 Mitogen Cona PHA one 54.3 3.21 56.7 63.5 2 27.0 2,3 32.1 47.6 3 59.2 4.3 148.4 123.0 four 73.0 2.7 129.2 117.0 5 24.8 2.2 49.7 43,2 6 11.7 1.34 19.7 23.1 7 4.7 2.1 139.7 126.8 8 2.9 1.7 24.6 21.3 9 5.3 0.56 44.7 53.1 10 27.6 0.46 163.1 143.9 eleven 57.2 0.1 68.7 65.3 12 10.9 5,6 56.9 72.1 13 62.3 6.25 67.2 57.4 fourteen 1.7 1.4 23.8 19.7 fifteen 33,4 1,2 36.0 34.5 16 7.2 10.7 77.8 67.3 17 98.7 11.7 72,2 78.1 eighteen 74.6 12.8 23.5 32,3 19 43.1 132.8 184.2 179.3 twenty 67.3 132.0 154.7 165.7

Table 3 Clinical status of tuberculosis patients identified using recombinant rCFP10 protein using IFN-γ analysis N p / p Tuberculosis infection Sensitivity (%) Specificity(%) one Infiltrative pulmonary tuberculosis 83-94 86-97 2 Focal pulmonary tuberculosis 82-93 90-94 3 Disseminated pulmonary tuberculosis 80-92 82-94 four Fibro-cavernous pulmonary tuberculosis 87-95 93-100

Claims (3)

1. Recombinant plasmid DNA rTB232 encoding the GST-CFP10 hybrid polypeptide with the properties of the mycobacterial antigen CFP10, with an average molecular weight of 3.4 MDa, has a size of 5257 bp and consists of the following elements:
4938 bp EcoGi-BamHI fragment of the vector plasmid pGEX-2T (Pharmacia Biotech) containing the β-lactamase gene, inducible tac promoter, lacI ^q internal gene encoding the lactose operon repressor protein, glutathione-3- gene fragment transferases from S. japonicum with a multiple site for cloning genes at the 3'end of this gene and a nucleotide sequence for a thrombin proteolysis site located in front of the multiple cloning site;
319 bp EcoRI-BamHI fragment containing the complete CFP10 protein gene flanked by restriction endonuclease sites EcoRI and BarnHI by amplification of the corresponding gene fragment with M. tuberculosis genomic DNA;
contains:
as a genetic marker, the β-lactamase gene that determines the resistance of the antibiotic ampicillin transformed with the plasmid pTV232 E. coli;
unique restriction sites: BamHI - 930/934, EcoRI - 1249/1253. 2. The recombinant bacterial strain Escherichia coli BL21 / pTB232, containing the recombinant plasmid DNA rTV232, according to claim 1, and deposited in the Microorganism Collection FGUN SSC VB "Vector" Rospotrebnadzor under the number B-1027, producer of the hybrid GST-CFP10 polypeptide with the properties of mycobacterial CFP10. 3. The recombinant GST-CFP10 polypeptide produced by the recombinant E. coli strain BL21 / pTB232 according to claim 2, contains a N-terminal polypeptide fragment of glutathione-S-transferase S.j. as a carrier protein. (226 a.a. with a molecular weight of 26.3 kDa) connected via the thrombin hydrolysis terminal site (LVPR ^ GS) to the C-terminal polypeptide fragment of the species-specific mycobacterial antigen CFP10 (100 a.a. with a molecular weight of 10.8 kDa) , and has a complete amino acid sequence (326 aa, 37.1 kDa) shown in FIG.

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