RU2458130C1 - RECOMBINANT PLASMID DNA pTB323 CODING HYBRID POLYPEPTIDE CST-DELTAMPT64 WITH PROPERTIES OF SPECIES-SPECIFIC MYCOBACTERIAL ANTIGEN MPT64 (MPB64), RECOMBINANT ESCHERICHIA COLI BACTERIAL STRAIN - PRODUCER OF HYBRID POLYPEPTIDE CST-DELTAMPT64 AND RECOMBINANT POLYPEPTIDE CST-DELTAMPT64 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: recombinant plasmid DNA pTB323 under the invention coding the hybrid polypeptide glutathione-8-transferase (GST) and a shorter version of the protein MPT64 (rΔMPT64), has an average molecular weight 3.6 MDa, size 5574 base pairs, consists of: a) EcoRI-BamHI-fragment of the vector plasmid pGEX-2T of size 4938 base pairs containing the β-lactamase gene inducing tac-promotor, the internal gene Iaclg coding the lactose operone repressor protein, a glutathione-5-transferase gene fragment from S. japonicum with a multiple sites of gene cloning (MSC) in 3'-terminal part of this gene and a nucleotide sequence coding a thrombine proteolysis site and found in front of the MSC; b) EcoRI-BamHI-fragment of 636 base pairs containing a truncated gene MPT64 flanked by EcoRI and BamHI restriction endonuclease sites and prepared by amplification of the gene-related fragment with genome DNA M. tuberculosis; c) a genetic marker - β-lactamase gene determining resistance of pTB323 plasmid transformed cells E. coli to the antibiotic ampicillin; d) unique restriction sites: BamHI - 930/934, EcoRI ~ 1566/1570. The recombinant bacterial strain Escherichia coli BL21/pTB323 - producer of hybrid polypeptide GST-ΔMPT64 with the properties of the mycobacterial antigen ΔMPT64 is deposited in the Collection of Microorganisms of Federal State Research Institution State Science Centre Vector, No. B-1028. The recombinant polypeptide GST-ΔMPT64 produced by the recombinant strain under the invention contains as a carrier protein the N-terminal polypeptide fragment glutathione-S-transferase S.j. (226 amino acid residues, 26.31 kDa) and has a complete amino acid sequence (431 amino acid residues, 48.76 kDa) presented in the description.

EFFECT: using the invention enables developing the high-purity polypeptide in the preparation amounts with the preserved immunogenic properties and provided separation of the target protein from the amino acid sequence of the carrier protein for studying of the immunogenic properties of the target protein.

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Description

The invention relates to biotechnology, in particular to genetic engineering, and allows microbiological synthesis using simplified technology to obtain a new hybrid GST-ΔMPT64 polypeptide with the properties of species-specific antigen protein Mycobacterium tuberculosis MPT64 (also corresponding to the homologous MPB64 protein Mycobacterium bovis earlier), which can be used diagnosis of tuberculosis infection caused by one of two types - Mycobacterium tuberculosis or Mycobacterium bovis .

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 persons vaccinated with BCG does not distinguish them from individuals infected with pathogenic mycobacteria that cause the development of tuberculosis in this test [2]. One of the reasons for obtaining false-positive results after the Mantoux reaction is associated with the manifestation of cross-reactivity to non-tuberculous mycobacteria. It is known that 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 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 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 human tuberculosis cross-react with a set of non-pathogenic antigens 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 (for example, CD4, CD8) [6]. For example, 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 production chain is a reflection of impaired immune system function [10].

γ-IFN analysis is based on a quantitative determination of the level of γ-IFN during lymphocyte stimulation of whole cell culture, incubated for 16-20 hours with protein antigens of M. tuberculosis and control antigens. As a result of stimulation of 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 show a lower percentage of vaccinees who have a positive result for the presence of tuberculosis infection, as determined by the skin 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 differentiating different phases of the development of tuberculosis infection in comparison with the results obtained using other, non-specific proteins, as well as a high level of T-cell response to species-specific antigens, which correlates with protective immunity in tuberculosis and with the risk of developing active tuberculosis.

The immunogenic protein MPB64, first identified by Harboe M. et al., Is known in the literature. [eleven]. As a native protein, MPB64 was found in Mycobacterium bovis BCG Tokyo culture fluid (filtrate). Later Andersen A.B. et al. [12] using monoclonal antibodies obtained on MPB64 protein, found a similar protein in the Mycobacterium tuberculosis H37Rv culture filtrate, which was designated as MPT64. It was also shown that the nucleotide sequence of both genes encoding these proteins coincides [13, 14]. The molecular weight of the purified native protein is 24 kDa. The main advantage of the MPT64 protein is that it is highly specific for the virulent species of tuberculosis pathogens ( M. tuberculosis, M. bovis, M. africanum ) [12] and in contrast to such well-known early proteins as ESAT-6 and CFP10, absent, as shown by Li H. et al., unfortunately, only in some genomes of BCG vaccine strains [15]. MPT64, along with major secretory proteins, such as ESAT-6 and CFP10, is one of the antigens that cause the development of a T-cell response. This process is accompanied by increased production of γ-IFN. The MPT64 protein gene is part of the 10.79 kbp RD2 genomic fragment present in the genomes of M. tuberculosis and M. bovis strains, including some M. bovis BCG strains [16]. Despite this, it can be used for diagnostic purposes, both in combination with other early proteins (ESAT-6, CFP10), and independently if applied to individuals previously vaccinated with vaccine strains that do not contain the mpt64 gene in their genome. It can also be used in relation to persons in those countries where the vaccination against tuberculosis is canceled, or to those vaccinated and sick (infected), based on the difference in the received immune response between only vaccinated and simultaneously vaccinated and sick (infected).

Isolation and purification of the MPT64 protein from the mycobacterial culture filtrate can be carried out using safe vaccine strains containing the MPB64 protein gene homologous to 100% MPT64. However, this procedure is expensive and complicated, and the yield of the target product 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.

A known genetic engineering method of producing a recombinant polypeptide with the properties of the mycobacterial antigen MPT64 used for diagnostic purposes [17, prototype]. In this case, the target protein is obtained as part of a recombinant fusion protein containing the target protein at the C-terminus in the form of a domain structure (complete and shortened — without the leader peptide — amino acid sequence variants), while the N-terminal amino acid sequence includes the malt-binding protein sequence (gene malE ) as a carrier protein (pMAL- p expression vector).

However, this design (prototype) does not contain a proteolysis site that allows you to separate the target protein from the amino acid sequence of the carrier protein for subsequent studies regarding the comparison of immunogenic properties with a protein approaching the native structure of the target or directly on the target.

The technical result of the invention is the creation of such a genetic construct and a recombinant strain of bacteria Escherichia coli based on it - a producer of a hybrid polypeptide with the properties of the species-specific mycobacterial antigen ΔMPT64 having such a design that would allow to produce the target highly purified polypeptide in preparative quantities while maintaining the immunogenic properties of the latter and the possibility of separation of the latter the target protein from the amino acid sequence of the carrier protein for immunoassay properties of the target protein.

This result is achieved by constructing pTB323 recombinant plasmid DNA encoding a chimeric polypeptide: glutathione S-transferase (GST) + a shortened version, without a leader peptide, MPT64 protein (hereinafter rΔMPT64). Moreover, the gene encoding the polypeptide with the properties of the mycobacterial antigen ΔMPT64 is in the same reading frame as the gene for glutathione S-transferase from Schistosoma japonicum (GST Sj) (Fig. 1A and B).

Recombinant plasmid DNA pTB323 (Fig. 1 A and B) with an average molecular weight of 3.6 MDa has a size of 5574 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;

- 636 bp EcoRI-BamHI fragment containing the truncated MPT64 gene flanked by restriction endonuclease sites EcoRI and BamHI, obtained by amplification of the corresponding gene fragment of M. tuberculosis genomic DNA;

and also contains:

- as a genetic marker, the β-lactamase gene, which determines the resistance of E. coli cells transformed with plasmid pTB323 to the antibiotic ampicillin;

- unique restriction sites: BamHI - 930/934, EcoRI - 1566/1570 (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 rΔMPT64 polypeptide allows the preparation of preparative amounts of the target protein using affinity chromatography on glutathione sepharose. These structural properties are important for the biotechnological cycle associated with the release 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 MPT64 mycobacterial antigen. When accumulated in a cell, rΔMPT64 practically (only to a small extent) does not undergo degradation. It also behaves steadily when stimulating a whole cell culture of blood in the process of diagnostic analysis. 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 rΔMPT64 polypeptide and the original one in the MAL-ΔMPT64 prototype is that the recombinant protein obtained as a result of expression is a single entity, which includes immunodominant species-specific structural epitopes of the native ΔMPT64 protein and does not have the functional activity of the GST fragment. The thrombin hydrolysis site located between the GST polypeptide fragments and ΔMPT64 (Fig. 2) allows, if necessary, the mycobacterial antigen ΔMPT64, free of the N-terminal GST polypeptide fragment, to be obtained. The recombinant ΔMPT64 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 strictly corresponding to the target protein ΔMPT64 - without a leader peptide, the first 23 amino acid residues full MPT64.

To obtain a bacterial strain producer of the chimeric protein rΔMPT64, competent E. coli BL21 cells are transformed with the constructed target plasmid pTB323. The resulting bacterial strain E. coli BL21 / pTB323 is characterized by the following features:

Morphological signs . According to morphological characteristics, the producer strain of the recombinant protein rΔMPT64 does not differ from the original E. coli BL21 strain that does not contain the target plasmid.

Cultural signs . According to cultural characteristics, the producer strain of the recombinant protein rΔMPT64 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 rΔMPT64 protein exhibit ampicillin resistance due to the presence of the target plasmid.

A significant difference between the strain E. coli BL21 / pТB323 is that it provides the synthesis of the chimeric rΔMPT64 polypeptide (GST-ΔMPT64) with the properties of the mycobacterial antigen ΔMPT64 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" Rospotrebnadzor under the number B-1028.

The chimeric rΔMPT64 polypeptide (GST-ΔMPT64) (Fig. 2) produced by the recombinant E. coli BL21 / pTB323 strain contains an N-terminal polypeptide fragment of glutathione-S-transferase Sj (226 aa with molecular weight 26.31 kDa; the calculation was performed using the Compute pI / Mw tool (Switzerland) program on-line (http://cn.expasy.org/tools/pi_tool.htmlhttp://cn.expasy.org/tools/ pi_tool.html)), connected via its terminal thrombin hydrolysis site (LVPR ^ GS) to the C-terminal polypeptide fragment of the species-specific mycobacterial antigen ΔMPT64 (205 a.a. with a molecular weight of 22.46 kDa; conducted using the Compute pI / Mw tool program), and has a complete amino acid sequence (431 aa, 48.76 kDa; calculation was performed using the Compute pI / Mw tool program) shown in FIG. 3.

The invention is illustrated by the following graphic materials.

FIG. 1 A. Scheme of the organization of the recombinant plasmid pTB323, where GST is the glutathione-S-transferase protein gene; ΔMPT64 — cloned fragment obtained by PCR amplification and including the truncated MPT64 protein gene; 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 resistance to ampicillin to transformed bacterial cells.

FIG. 1 B. Restriction map of the target plasmid pТB323. Image of the construction of plasmid pTB323 (5574 bp). a - fusion recombinant protein GST-ΔMPT64 size 431 a.o. Images are built using NEBcutter V2.0 (UK) ( http://tools.neb.com/NEBcutter2/index.php )

FIG. 2. Docking sites when the Δ mpt64 gene of plasmid pTB323 is introduced into the glutathione-S-transferase ( gst ) gene in comparison with the same region 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.

FIG. 3. The complete nucleotide and amino acid sequence of the recombinant GST-ΔMPT64 polypeptide, where LVPR ^ GS is the 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 pTB323.

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}.

Genomic DNA of M. tuberculosis H37Rv 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 ΔMPT64 protein is amplified using primers:

5'-CG GGATCC GCGCCCAAGACCTACTG-3 '(direct primer for the mpt 64 gene);

5'-CG GAATTC GTCCTCGCGAGTCTAGG-3 '(reverse primer for the mpt 64 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 - 3 min; 10 cycles [94 ° C - 30 s, 45 ° C - 30 s, 72 ° C - 50 s], 35 cycles [94 ° C - 30 s, 60 ° C - 30 s, 72 ° C - 50 s], 72 ° C - 3 min. The complete nucleotide sequence of the cloned gene ( mpt64 ) for selection of the nucleotide sequence of the primers was extracted from the database (DB) of the Pasteur Institute ( http://genolist.pasteur.fr/TubercuList/ ) (France). The primer melting temperatures are selected based on the extended nucleotide sequence obtained from the database, including the truncated Δ mpt64 gene, using the NTI Suite 8. The primers are annealed in the first 10 cycles at a low annealing temperature (45 ° C), since the 5'-terminal nucleotide sequence of 8 n.a., in the structure of which the restriction site is laid (in this case BamHI or EcoRI), it cannot form a perfect hybrid duplex with the DNA matrix ( M. tuberculosis ) at the initial moment of PCR after a longer 3 ' -end The second part of the 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 temperatures for the primers were calculated using the NTI Suite 8 software or the Oligonucleotide Properties Calculator (http://www.basic.northwestern.edu/biotools/oligocalc.html) (USA) 5'-end sequence of 8 n.a. 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 (Sibenzym, Novosibirsk, Russia). The resulting PCR fragment (amplicon) of 646 bp in length purified by reprecipitation with ethanol in the presence of sodium acetate, pH 4.8, and tRNA as a carrier [18]. The purified amplicon preparation was dissolved in 80 μl of TE buffer and then co-hydrolyzed in EcoRI buffer (Sibenzyme) with BamHI and EcoRI restriction endonucleases 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) is 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 cells of E. coli strain JM103 prepared according to the method using a solution of calcium chloride [18], which are seeded on plates with agar medium containing 50 μg / ml ampicillin. 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. In this case, clones containing the insertion of the truncated Δ mpt64 gene 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 806 bp, which exceeds the length of the cloned fragment, how 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 pTB323. The plasmid DNA scheme is shown in FIG. 1 A and 1 B.

Example 2. Obtaining a producer strain, the cultivation of cells which produces the chimeric protein GST-ΔMPT64.

Cell transformation of E. coli strain BL21 is carried out by introducing the DNA of the target plasmid pTB323 into competent cells prepared according to the method using a solution of calcium chloride [18], after which they are plated on plates with agar medium containing ampicillin at a concentration of 50 μg / ml. 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. Δ mpt64 gene expression 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 rΔMPT64, the amount and concentration of which is estimated by the electrophoretic picture (in our case, the cultivation parameters are 37 ° C and 1.0 mM IPTG) .

Example 3. Isolation of the recombinant protein GST-ΔMPT64.

Cells of E. coli strain BL21 transformed with pTB323 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 a density of D ₅₅₀ = 0.8 for 3 hours. Next, expression of the target protein GST-ΔMPT64 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-ΔMPT64 protein is 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 a BL21 / pTB323 strain in the E. coli cell culture induced when processing cell sediments with a GST-ΔMPT64 lysis buffer protein with a molecular weight of about 48-50 kDa, 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 (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 min at 0 ° C and the soluble protein fraction is separated from the debris by centrifugation at 12000g 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-ΔMPT64 protein. The mixture was then transferred to a microcolumn by repeated centrifugation at 600 g, the column was washed several times with FSB buffer and the recombinant 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. 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 RPMI 1640 medium with L-glutamine. Thoroughly mix and make antigen GST-ΔMPT64 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 produced by BD Biosciences in various concentrations (from 300-150-75-37.5-18) are added to the control wells of a control plate with sorbed monoclonal antibodies to gamma interferon manufactured by BD Biosciences. 8-9.4-4.7 pg / ml). In the remaining wells of the tablets, 100 μl of the studied whole blood samples are added after stimulation, placed in a shaker and incubated for 2 hours at room temperature. After incubation, the wells of the plates 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 distilled water. 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 stopped by adding to each well of the tablet 50 μl of a stop reagent (0.9 N solution of H ₂ SO ₄ ). 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. The results are calculated from the average OD value in three wells (ρ <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 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 tablets (Nunc, MaxiSorb, USA) containing 150 μl / well of complete RPMI 1640 medium manufactured by the State Scientific Center of the World Bank "Vector". Then, chimeric antigen GST-ΔMPT64 at a concentration of 5-10 μg / ml is added to each well of the plate; mitogen (Con A or PHA) was added to the control wells of the plate 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 are added to the control wells of the plates with sorbed monoclonal antibodies to gamma-interferon. In the remaining wells of the tablets, 100 μl of the investigated blood plasma samples are added after stimulation. Placed in a shaker and incubated for 2 hours at room temperature. After incubation, the wells of the plate 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 plates are placed in a dark place and kept at room temperature for 30 minutes. The reaction is stopped by adding to each well of the tablet 50 μl of a stop reagent (0.9 N solution of H ₂ SO ₄ ). 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 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. 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 The sorption mAb to IFN-γ production BD OptEIA Reagent performed using a 0.1 M carbonate buffer, pH 9.6 for 16 hours at 4 ^{° C} on plates (Nunc, MaxiSorb, USA). After washing the plates with washing buffer solution (FSB, pH 7.0) 3 times 300 μl / well, the surface of the wells is blocked with FSB blocking buffer solution, pH 7.0, containing 10% fetal serum, for 60 min at room temperature, after which carry out the washing of the tablets, 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). Mix thoroughly and make a solution of the GST-ΔMPT64 antigen in RPMI 1640 with 0.2 mM glutamine at a concentration of 10 μg / ml per well. A PHA mitogen is added to wells A-1, B-1, and C-1, and 100 μl of dilution buffer solution are added to wells D-1, E-1 (zero control). 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 FSB-T buffer solution and 5 × 300 μl with distilled water. To construct a calibration graph during the IFN-γ analysis, incubation is preliminarily performed with standard IFN-γ calibration samples. For this, 100 μl of standard samples of gamma-interferon produced by BD Biosciences in various concentrations (from 300-150-75-37.5-18.8-9, are added to the control wells of plates with sorbed monoclonal antibodies to gamma interferon manufactured by BD Biosciences) 4-4.7 pg / ml) or 100 μl of standard calibration samples containing IFN-γ (2000-1000-500-300-100-50-0 pg / 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 of the plates are 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 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 tablet. The tablets are placed in a dark place and kept at room temperature for 30 minutes. The reaction is stopped by adding 50 μl of a stop reagent (0.9 N H ₂ SO ₄ solution) to each well of the plate and the optical density 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 contingent does not exceed the limit values -> 300 pg / ml IFN-γ, corresponding to the risk group or the group of patients with tuberculosis. These data indicate the specificity of the method.

Table 1

IFN-γ analysis of tuberculosis during rMPT-64 fusion protein stimulation in patients with active pulmonary tuberculosis

The patients IFN-γ (pg / ml) PPD rΔMPT-64 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.

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).

Table 2.

IFN-γ analysis of pulmonary tuberculosis when stimulated with the fusion protein rΔMPT64 and mitogens

The patients IFN-γ (IU / ml) PPD rΔMPT-64 Mitogen Cona PHA one
2
3
four
5
6
7
8
9
10
eleven
12
13
fourteen
fifteen
16
17
eighteen
19
twenty 54.3
27.0
59.2
73.0
24.8
11.7
4.7
2.9
5.3
27.6
57.2
10.9
62.3
1.7
33,4
7.2
98.7
74.6
43.1
67.3 4.23
4.1
3,1
1.3
1.9
0.98
1.12
1,2
0.57
0.23
0.12
3,7
6.1
0.3
0.78
9.75
8.9
11.7
111.2
103,4 56.7
32.1
148.4
129.2
49.7
19.7
139.7
24.6
44.7
163.1
68.7
56.9
67.2
23.8
36.0
77.8
72,2
23.5
184.2
154.7 63.5
47.6
123.0
117.0
43,2
23.1
126.8
21.3
53.1
143.9
65.3
72.1
57.4
19.7
34.5
67.3
78.1
32,3
179.3
165.7

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 clinical status of patients with various forms of tuberculosis infection identified using the rΔMPT64 fusion protein is shown in Table 3. For another group of patients with tuberculosis infection, IFN-γ production results were expressed in IU / ml using a calibration graph.

Table 3.

Clinical status of tuberculosis patients identified using recombinant protein rMPT-64 using IFN-γ analysis

No.
pp Tuberculosis infection Sensitivity (%) Specificity
(%) one Infiltrative pulmonary tuberculosis 73-84 74-97 2 Focal pulmonary tuberculosis 72-83 80-94 3 Disseminated pulmonary tuberculosis 70-82 82-94 four Fibrous cavernous pulmonary tuberculosis 77-85 83-100

Table 4 presents the results of a comparative study of the specificity and sensitivity of the recombinant protein rΔMPT64 in comparison with PPD-tuberculin during stimulation of whole cell blood culture. The specificity of the recombinant protein in the IFN-γ analysis for the detection of tuberculosis is higher than the PPD-tuberculin.

Some forms of tuberculosis (acutely progressive) cannot be detected during preventive examinations. Patients with such processes go mainly to 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).

Claims (3)

1. Recombinant plasmid DNA rTB323 encoding a hybrid polypeptide: glutathione S-transferase (GST) and a shortened version of the protein MPT64 (rΔMPT64), has an average molecular weight of 3.6 MDa, size 5574 bp and consists of the following elements:
- EcoRI-BamHI - fragment of the vector plasmid pGEX-2T with a size of 4938 bp containing the β-lactamase gene, inducible tac promoter, internal lacI ^q gene encoding the repressor protein of the lactose operon, glutathione S-transferase gene fragment 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 located in front of MSCs;
- EcoRI-BamHI - fragment 636 bp in size, containing the truncated MPRI 64 gene flanked by sites for restriction endonucleases EcoRI and BamHI, obtained by amplification of the corresponding fragment with M. tuberculosis genomic DNA;
and contains:
as a genetic marker, the β-lactamase gene that determines the resistance of E. coli cells transformed with plasmid rTB323 to the antibiotic ampicillin;
- Unique restriction sites: BamHI - 930/934, EcoRI - 1566/1570. 2. Recombinant bacterial strain Escherichia coli BL21 / pTB323-producer of the hybrid polypeptide GST-ΔMPT64 with the properties of the mycobacterial antigen ΔMPT64, containing the recombinant plasmid DNA rTB323 according to claim 1 and deposited in the Microorganism Collection FGUN GNTS VB Supervisory Public Health Center No. 10 No. 10, Vector. 3. The recombinant GST-ΔMPT64 polypeptide produced by the recombinant E. coli BL21 / pTB323 strain 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 26.31 kDa) connected through its terminal site of thrombin hydrolysis (LVPR ^ GS) to the C-terminal polypeptide fragment of the species-specific mycobacterial antigen ΔMPT64 (205 a.a. with a molecular weight of 22.46 kDa ) and has a complete amino acid sequence (431 aa with a molecular weight of 48.76 kDa) shown in Fig.3.

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