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

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 (LVPRGS) 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

 

The invention relates to biotechnology, in particular genetic engineering, and allows you to get the microbiological synthesis of simplified technology new hybrid polypeptide GST-CFP10 with properties of species-specific protein antigen of Mycobacterium tuberculosis CFP10, 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 skin reactions at 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 results in 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 tubercul is for the greatest 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, fast production procedures, high sensitivity and specificity among infected individuals [3]. Serological methods are very diverse. The sensitivity and specificity of tests that use technology enzyme-linked immunosorbent assay (ELISA), varies in a rather wide range and is 40-85% and 67-100%, respectively. On the one hand, this is due to the fact that often indicators of humoral immune response in tuberculosis has been reduced and this, in turn, leads to the fact that antibodies obtained in the human body, to virulent the m mycobacteria, causing tuberculosis in humans, cross-react with a set of non-pathogenic antigens of mycobacteria and many agents of other diseases. On the other hand, antimycobacterial and hormonal therapy have a suppressive effect on the production of antibodies in tuberculosis. The last factor is essential when identifying markers of humoral immunity in TB patients, since the reduced level of antibodies entails a decline in the share of positive results in the ELISA, which 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 proteins of other species of the genus Mycobacterium and even other genera of bacteria. A number of proteins of the culture filtrate, secreted by mycobacteria M. tuberculosis and have immunogenic 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 effectors (e.g., CD, CD8) [6]. For example, SV-T-cells produce immune interferon (IFN-γ, IFN-γ), which is a stimulator in the infected organism antimycobacterial macrophages, as shown in mouse models [7], as well as an activator of macrophages person in suppressing infection .tuberculosis [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].

High level production of 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 of 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 cells in the blood is rapid secretion of C is Takenov, 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 serves as a 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) .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 revealed a lower percentage vaccinated with a positive result for the presence of tuberculosis infection than the skin test data. 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 various phases of development of tuberculosis is infection in contrast to other proteins .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 against M. tuberculosis and the risk of developing active TB.

Known CFP10 protein, first identified Berthet FX with co-authors [11], as it is highly specific for virulent species of causative agents of tuberculosis. As native CFP10 protein was detected in the cytosolic fraction and the cell wall of mycobacteria, as well as in the culture fluid. The molecular mass of the purified native protein of 14 kDa. The main advantage of this protein in the fact that in the genome of BCG vaccine strains lacking the nucleotide sequence encoding it [12]. CFP10 is the main antigen, causing the development of early T-cell response. This process is accompanied by increased production of γ-IFN. Gene CFP10 protein is part of the genomic fragment RD1 size 9.5 thousand gel present in the genomes of strains of M. tuberculosis and M. bovis, including in laboratory strains of M. tuberculosis (H37Rv, H37Ra). But this nucleotide sequence (RD1-region) is missing in all vaccine strains of M. bovis BCG, and this means that CFP10 also missing in vaccine preparations that can be used in diagnostic purposes [12].

Isolation and purification of CFP10 protein from the culture f is ltrate mycobacteria is expensive and complicated procedure, the product yield is extremely low. This is why the technique of recombinant DNA to obtain the protein for practical diagnostic purposes is economically feasible.

Known genetic engineering method of obtaining a recombinant polypeptide with properties of mycobacterial antigen CFP10 used for diagnostic purposes [11]. In this case, get the target protein with a 6 his-tag (6 His) amino acid residues at N-end.

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

- hybrid protein containing the full CFP10 protein from M. tuberculosis, merged with a complete protein genes esat6 of M.tuberculosis through a linker amino acid sequence;

recombinant plasmid DNA pTBD16 for expression of the hybrid protein CFP10-genes esat6.

a strain of E. coli DLT1270 transformed the obtained recombinant plasmid DNA pTBD16 producing hybrid CFP10 protein-genes esat6 [14].

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 CFP10 the latter undergoes significant degradation, which affects its immunogenic properties.

The technical result is m invention is to provide a recombinant strain of Escherichia coli bacteria - producer of hybrid polypeptide with properties of species-specific mycobacterial antigen CFP10 having such a construction that would allow us to develop highly targeted polypeptide in preparative quantities while maintaining the immunogenic properties of the latter.

This result is achieved by constructing recombinant plasmid DNA RTV that encodes a chimeric polypeptide: glutathione-S-transferase (GST)+CFP10 (hereinafter rCFP10). When this gene that encodes a polypeptide with properties of mycobacterial antigen CFP10, is in the same reading frame with the gene glutathione S-transferase from Schistosoma japonicum (GST unlimited company.) (Figa and B).

Recombinant plasmid DNA RTV (Figa and 1B) with an average molecular weight of 3.4 MDA has a size 5257 P.N. (design image plasmids, restrictee card size calculations performed using NEBcutter V2.0 (UK) in on-line mode (http://tools.neb.com/NEBcutter2/index.php)) and consists of the following elements:

- EcoRI-BamHI fragment of the vector plasmid pGEX-2T (Pharmacia Biotech) size 4938 gel, containing the gene for β-lactamase induced tac-promoter, lacI geneqencoding the protein is a repressor of Lac operon, a gene fragment glutathione-S-transferase from S. japonicum with multiple site for cloning (MSC) genes in the 3'end of this gene and a nucleotide sequence that encodes a SAI is proteolysis by thrombin and located in front GMT;

- EcoRI-BamHI fragment size 319 gel containing flanked by sites for the restriction endonucleases EcoRI and BamHI full gene CFP10 protein, obtained by amplification of the corresponding gene fragment from genomic DNA of M. tuberculosis;

contains:

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

unique restriction sites: BamHI -930/934, EcoRI -1249/1253(indicated by the position of the splitting of the appropriate restriction endonucleases on both circuits, numbering when this is carried out on the same chain).

The presence of recombinant polypeptide rCFP10 GST-fragment allows to develop preparative quantities of the target protein using affinity chromatography. These design features important for biotechnological cycle in the secretion of the recombinant protein. It is 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 CFP10. In the form of a recombinant fusion protein rCFP10 not undergo degradation and is significantly more stable similar, containing the C-terminal part of ESAT-6, previously obtained (patent RF №2282661). Apparently, the presence of GST-fragment in the composition hiberno what about the protein helps to protect it from the effects of enzymes blood upon stimulation of whole cell cultures of blood in the process of diagnostic analysis. The prolongation of its action upon stimulation of whole blood or mononuclear cells in 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 CFP10 is that the recombinant protein, the resulting expression is a single education, including species-specific immunodominant structural epitopes of the native protein CFP10 and not having a functional activity of GST-fragment. Located between the polypeptide fragments of GST and CFP10 (2) site of thrombin hydrolysis allows you to get mycobacterial antigen CFP10-free N-terminal polypeptide fragment of the GST. Recombinant CFP10 antigen on the N-end after cleavage by thrombin will contain only two additional amino acid residue glycine is N-terminal, serine - second amino acid residue, followed by amino acid sequence, strictly corresponding target protein CFP10.

To obtain a bacterial strain-producer chimeric protein rCFP10 competent cells of E. coli BL21 transformed constructed target plasmid RTV. Received the Tamm bacteria E. coli BL21/pTB232 characterized by the following features:

Morphological features. The morphological characteristics of the strain-producer of recombinant protein rCFP10 not differ from the original E. coli strain BL21, not containing target plasmid.

Cultural characteristics. Cultural characteristics of strain-producer of recombinant protein rCFP10 not differ from the original E. coli strain BL21, not containing target plasmid.

Resistance to antibiotics. Cells of strain-producer of recombinant protein rCFP10 are resistant to ampicillin due to the presence of the target plasmid.

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

The resulting strain was deposited in the culture Collection of microorganisms (CCM) fsri SRC VB "Vector" of Rospotrebnadzor under number B-1027.

Chimeric polypeptide rCFP10 (GST-CFP10) (Figure 2), produced by recombinant strain BL21/pTB232 E. coli, contains as a protein carrier N-terminal polypeptide fragment of glutathione-S-transferase unlimited company. (226 S.A. with a molecular mass of 26.3 kDa; the calculation is performed using the Compute pl/Mw tool (Switzerland) in on-line mode (http://cn.expasy.org/tools/pi tool.html)connected through its terminal site of thrombin hydrolysis (LVPR^GS) with C-terminal polypeptide fragment of the species-specific mycobacteria inogo CFP10 antigen (100 S.A. with the molecular weight is 10.8 kDa; the calculation is performed using the Compute pl/Mw tool), and has a complete amino acid sequence (326 S.A., 37,1 kDa; the calculation is performed using the Compute pl/Mw tool, shown in Figure 3.

The invention is illustrated in the following graphics.

Figa. Organization of the recombinant plasmids RTW, where GST - gene protein glutathione-3-transferase; CFP10 - cloned fragment obtained by amplification using PCR and including full gene CFP10 protein; unique BamHI and EcoRI sites of the restriction endonucleases used to create this design; ptac - synthetic promoter present in the vector (pGEX-2T) and the recombinant plasmids; ApRgene β-lactamase (bla gene)providing a transformed bacterial cells resistant to ampicillin.

Figb. Restriction map of a target plasmid RTV. The image construction plasmids RTV (5257 P.N.). b - fused recombinant protein GST-CFP10 size 326 S.A. Image built using NEBcutter V2.0 (UK) (http://tools.neb.com/NEBcutter2/index.php) [Vincze, T., pó sfai, J. and Roberts, R.J. NEBcutter: a program to cleave DNA with restriction enzymes. Nucleic Acids Res. 31: 3688-3691 (2003)], is available online.

Figure 2. The parts of the coupling when the introduction of gene esxB plasmids RTW in gene glutathione S-transferase (gst) in comparison with the similar plot in the original (vector) PLA is the MFA pGEX-2T. NP - nucleotide sequence, up - amino acid sequence. The first codon of the protein is shown after installation of the nucleotide sequence of the site of proteolysis by thrombin, the stop codon at the end of the gene-insertion underlined.

Figa and B. the Complete nucleotide and amino acid sequence of the recombinant polypeptide GST-CFP10, where- amino acid sequence of the site of recognition and hydrolysis of the protease thrombin (highlighted).

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

Example 1. Construction of recombinant plasmids RTW

Used as a vector plasmid pGEX-2T (Pharmacia) and strains: E. coli JM103 {Δ(lac-proAB), thi, strA, supE, endA, sbcB, hsdR-, [F' traD36, proAB, lacIq, ZΔM15]}; BL21 E. coli In {F-, dcm, ompT, hsdS (rB- mB-), gal}.

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

5'-GAGGATCCATGGCAGAGATGAAGACC-3' (forward primer for gene esxB); 5'-GCGAATTCTATTAGCGGGTCAGAAGC-3' (reverse primer for gene esxS). PCR is carried out in a volume of 50 μl on the amplifier "Terzic" ("DNA technologist who I am", Moscow) with the temperature-time profile: 94°C - 3 min; 5 cycles of [94°C 30 sec, 45°C - 20 s, 72°C - 40], 30 cycles of [94°C 30 sec, 58°C for 20 s, 72°C - 40], 72°C - 3 minutes Complete nucleotide sequence of the cloned gene (esxB) for the selection of primary nucleotide sequences of the primers were extracted from the database (DB) of the Institut Pasteur (http://genolist.pasteur.fr/TubercuList/) (France). The melting temperature of the primers selected on the basis of the obtained from the database of the extended nucleotide sequence comprising the full gene esxB, using NTI Suite 8. Annealing of the primers in the first 5 cycles carried out at a low temperature annealing (45°C), because the 5'-end nucleotide sequence of 8 concentration, the structure of which the restriction site (in this case, BamHI or EcoRI), cannot form the DNA-matrix (M. tuberculosis) is a perfect hybrid duplex in the initial flow PCR after a long 3'-end portion of the primer formed it. In this case, the calculation of the melting temperature of each primer are only fully hybridization sequence, discarding the first 8 determination of the structure of the primer. Calculations of the melting temperature for primers was performed using the program NTI Suite 8 or Oligonucleotide Properties Calculator (http://www.basic.northwestern.edu/biotools/oligocalc.html) (USA). 5'-end sequence of 8 concentration is complementary to the primary nucleotide is the first sequence of each primer and is designed to create 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; 4 deoxynucleosides - dATP, DSTF, dCTP and TTP - with the final concentration of 0.2 mm of each; the primers with the concentration of each of 0.15-0.25 μm and 5 units of Taq-DNA-polymerase ("Simanim", Novosibirsk, Russia). The output is about 2 mkg (8 pmol) PCR fragment. The obtained PCR fragment (amplicon) length 329 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 restrictly fragment 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) using the restriction endonucleases BamHI and EcoRI, also containing 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 PE is UNOSAT in thermostat, where was incubated overnight at 16°C. an Aliquot ligase mixture (1-2 µl) was used to transform competent cells of E. coli strain JM103, prepared according to 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°C over 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 gene esxB (cfp10), after separation of amplificata electrophoresis in agarose gel (1.2%in the presence of ethidium bromide to show painted under UV light strip corresponding to the length of 489 BP, which exceeds the length of the cloned fragment, this fragment contains a portion of the nucleotide sequence of the source vector molecule. 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 RTV. Scheme of plasmid DNA presents on Figa and 1B.

Example 2. Obtaining strain-p is ducenta, when culturing cells which are products of chimeric protein GST-CFP10

Transformation of cells of the strain E. coli BL21 carried out by the introduction of DNA target plasmids RTW 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°C over 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 YT liquid medium with ampicillin at a concentration of 50 μg/ml for the night cell cultures and incubated at 37°C without swing. The next day seeded overnight culture of each clone in the ratio of 1:100 2 ml of fresh medium YT with ampicillin at a concentration of 50 μg/ml and incubated at 37°C with a swing speed of 160 rpm./min prior density of D550=0.5 to 0.8. Gene expression esxB induce the addition of isopropyl-β-D-galactopyranoside (IPTG) to a final concentration of 0.5-1.0 mm, incubated at 37°C under intensive aeration (about 160-170./min) for 3-5 h, and then select an aliquot of cell suspension of each clone and derived from cell culture lysates analyzed by electrophoresis in 10%SDS page under denaturing conditions according to laemmli's method. The final selection of clones was carried out by the ability of culturegeek each individual clone to produce the maximum possible amounts (after induction) recombinant protein rCFP10, the amount and concentration of which is judged by electrophoretic painting (in this case 37°C and 1.0 mm IPTG).

Example 3. Selection of the recombinant protein GST-CFP10

Cells of strain BL21 E. coli transformed with plasmid RTV, grown in LB medium with ampicillin (50 μg/ml) overnight at 37°C. the overnight culture (1:100) seeded in 150 ml of LB medium with ampicillin (50 μg/ml) and pokasivaut to the density D550=0.8 V for 3 hours Then induce the expression of target protein GST-CFP10 adding IPTG to a concentration of 1.0 mm. Induced cells grow at 37°C for 5 h, after which the cell culture is cooled to 0°C, and cells harvested by centrifugation at 5000g for 15 min at 4°C. the Expression of the target protein GST-CFP10 analyzed 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 cell culture of E. coli strain BL21/pTB232 in the processing of cellular precipitation lytic buffer GST-CFP10 protein with a molecular weight of about 37 kDa, which corresponds to the calculated molecular mass. In control lysates of cells E. coli BL21 and BL21/pGEX-2T E. coli protein of this molecular weight is missing (according to electrophoresis). This protein is colored in immunoblotting with monoclonal antibodies to protein-media GST.

To give the further analyses 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 KH2PO4pH of 7.3, 1 mm PMSF (protease inhibitor), and destroy ultrasound in an ultrasonic disintegrator (Ultrasonic processor Cole Parmer) 3×30 c with an interval of 1 min between treatments at an amplitude of 50 A. To ultrasonic desintegrator add Triton X-100 to 1%, incubated for 30 min at 0°C and separate the fraction of soluble proteins from debris by centrifugation at 12000g 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 agitation at room temperature for affinity binding protein GST-CFP10. The mixture is then transferred into microcolony by repeated centrifugation at 600g, washed several times with column FSB (PBS)buffer and perform the elution of the recombinant target protein buffer for elution containing 10 mm restored glutathione in 50 mm Tris-HCl, pH 8.0. The result is a purified preparation of recombinant protein with a concentration of 1.5 mg/ml

Example 4. Holding IFN-γ-analysis

1st stage (stimulation of whole cell cultures of 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 RPMI medium 1640 with L-g is aluminum. Mix thoroughly and make the antigen GST-CFP10 at 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 tablets 24-48 h at 37°C in a humid atmosphere containing 5% CO2

2nd stage. To conduct IFN-γ assays in 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). In the remaining wells tablet make 100 μl samples of whole blood after stimulation, placed in a shaker and incubated for 2 h at room temperature. After incubation, the wells 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 production from BD Biosciences and incubated for 60 min at room temperature. After incubation wash the tablets 3 times with the buffer solution FSB-T, then in 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 tablets washed with 5 the AZ wash buffer (FSB-T) and 3 times H 2O distilled. Then bring in each well of the tablet 100 μl of a solution of TMB (3,3',5,5'-tetramethylbenzidine), put the tablets 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 (PG/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 (p<0,05). The Cutoff values for IFN-γ>300 PG is considered positive.

Example 5. Mononuclear cells whole cell cultures of blood (2×105cells/well)isolated by standard procedure using gradient centrifugation in the presence of reagents Ficoll-Hipac, 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 the each well of the tablet make chimeric antigen GST-CFP10 at 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 tablets 16-24 h at 37°C in a humid atmosphere containing 5% CO2.

To conduct IFN-γ assays in 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 tablets 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 wash the tablets 3 times with the buffer solution FSB-T, then in each well of the tablet add 100 ál of conjugate avidin-horseradish peroxidase in the working dilution and incubated for 60 min at room temperature. After incubation tablets 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 tablets in the dark place and videri is up at room temperature 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 (p<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 9.6 for 16 h at 4°C on plates (Nunc, MaxiSorb, USA). After flashing the tablet wash buffer (FSB, pH 7.0) 3 times with 300 µl/well hold the lock on the surface of the hole blocking buffer solution FSB, pH 7.0, containing 10% fetal serum for 60 min at room temperature, followed by washing the tablet as described above.

The prepared tablet with sorbed µa to IFN-γ used in the conduct of IFN-γ analysis. For this purpose, in each well of the tablet make 10 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-CFP10 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 mixed, incubated tablets 24-48 h at 37°C in a humid atmosphere containing 5% CO2when mixing. After incubation tablets washed with 5×300 μl/well of distilled water and 5×300 μl 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 300-150-75-37,5-18,8-9,4-4,7 PG/ml) production BD Biosciences or 100 ál of standard calibration samples containing IFN-γ (2000-1000-500-300-100-50-0 PG/ml) and control sample (330 PG/ml IFN-γ) production "Vector-best". Incubated according to the instructions for use of the kits. After incubation the strips tablet washed 3 times with wash buffer rastv the rum (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 tablets 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 tablets washed 5 times with wash buffer (FSB-T), 3 times with distilled water, then bring to each well of the tablet 100 μl of a solution of TMB (3,3',5,5'-tetramethylbenzidine). The tablets are placed in a 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) 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 get constructed 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 the WMD 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 using IFN-γ-analysis, as well as among healthy population. The conducted studies show that received recombinant protein allows 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), the 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 a hybrid protein rCFP10 presented in table 3. Table 4 presents the results of a comparative study of the specificity and sensitivity of the recombinant protein rCFP10 compared to PPD-tuberculin stimulation of whole cell cultures of blood. The specificity of the recombinant protein in IFN-γ-analysis of TB is higher than the PPD tuberculin.

For another group of patients with tuberculosis infection results in the production of IFN-γ was expressed in IU/ml using the calibration graph. The analytical sensitivity of the test was 1.2-1, IU/ml IFN-γ for negative (zero) control sample from the blood plasma of the subjects (results are presented in table 2).

Some forms of TB (acute progressive) may not be detected during the screening. Patients with such processes fall mainly in the General hospital, where their diagnosis is often 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).

Table 1
IFN-γ-analysis of tuberculosis upon stimulation with PPD, a hybrid protein rCFP10 and mitogens in patients with active form of tuberculosis
PatientsIFN-γ (PG/ml)
PPDrCFP10Mitogen
ConARNA
214100 84567004782
223650198573245421
2328505545303218
244870320073006710
255110275596508971
266200284978618764
271732057801234414230
2839806748514352
29583042101987021340
301035074001830017235
31212004151654014530
32573020651234713247
33670572345032410
341534038751765018930
352319640985
36745821902341028750
371048062401934018745
38613017454500 29560

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

Table 2
IFN-γ-analysis of pulmonary tuberculosis upon stimulation with PPD, a hybrid protein rCFP10 and mitogen -
PatientsIFN-γ (IU/ml)
PPDrCFP10Mitogen
ConARNA
154,33,2156,763,5
227,02,332,147,6
359,24,3of 148.4123,0
473,02,712,2 117,0
524,82,249,743,2
611,7of 1.3419,723,1
7the 4.72,1139,7to 126.8
82,91,724,621,3
95,30,5644,753,1
1027,60,46to 163.1143,9
1157,20,168,765,3
1210,95,656,972,1
1362,3 6,2567,257,4
141,71,423,819,7
15the 33.41,236,034,5
167,210,777,867,3
1798,711,772,278,1
1874,612,823,532,3
19to 43.1132,8184,2179,3
2067,3132,0154,7165,7

Table 3
Clinical status of patients is tuberkuleza, identified using recombinant protein rCFP10 using IFN-γ-analysis
N p/pTB infectionSensitivity (%)Specificity(%)
1Infiltrative tuberculosis of the lungs83-9486-97
2Focal pulmonary TB82-9390-94
3Disseminated tuberculosis80-9282-94
4Fibrous-cavernous lung tuberculosis87-9593-100

1. Recombinant plasmid DNA RTV encoding a hybrid polypeptide GST-CFP10 properties of mycobacterial antigen CFP10, with an average molecular weight of 3.4 MDA has a size 5257 P.N. and consists of the following elements:
EcoRI-BamHI fragment of the vector plasmid pGEX-2T (Pharmacia Biotech) size 4938 gel, containing the gene for β-lactamase is, inducible tac-promoter, lacI geneqencoding the protein is a repressor of Lac operon, a gene fragment glutathione-3-transferase of S.japonicum with multiple site for cloning genes in the 3'end of this gene and the nucleotide sequence for the site of proteolysis by thrombin, which is located in front of multiple site for cloning;
EcoRI-BamHI fragment size 319 gel containing flanked by sites for the restriction endonucleases EcoRI and BarnHI full gene CFP10 protein, obtained by amplification of the corresponding gene fragment from genomic DNA .tuberculosis;
contains:
as a genetic marker gene β-lactamase determining the stability of the transformed plasmid RTW of E.coli cells to the antibiotic ampicillin;
unique restriction sites: BamHI - 930/934, EcoRI - 1249/1253.

2. Recombinant bacterial strain Escherichia coli BL21/pTB232 containing recombinant plasmid DNA RTV according to claim 1, and deposited in the Collection of microorganisms fsri SRC VB "Vector" of Rospotrebnadzor under number B-1027, producer of hybrid polypeptide GST-CFP10 properties of mycobacterial antigen CFP10.

3. Recombinant polypeptide GST-CFP10 produced by recombinant strain E. coli BL21/pTB232, according to claim 2, contains as a protein carrier N-terminal polypeptide fragment of glutathione-S-transferase unlimited company. (226 S.A. with molecular masses, the th of 26.3 kDa), connected through terminal site of hydrolysis by thrombin (LVPR^GS) with C-terminal polypeptide fragment species-specific mycobacterial antigen CFP10 (100 S.A. with the molecular weight is 10.8 kDa), and has a complete amino acid sequence (326 S.A., 37,1 kDa), is shown in figure 3.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention can be used in manufacturing of vaccines for Streptococcus pyogenes - streptococci of group A (SGA) and Streptococcus agalactiae - streptococci of group B (SGB). Substance of the invention involves development of recombinant DNA pB1 derived from PCR with using chromosomal DNA of strain 090R Ia of serotype SGB, primers Pb1 and Pb2 and following cloning with using expression plasmid pQE-30 in E coli M15. Recombinant DNA pB1 codes recombinant protein PB1 expressing protective properties in relation to specified streptococci which has no enzymatic activity and causes synthesis of anti-Pb1 antibodies expressing protective properties in relation to Streptococcus pyogenes and Streptococcus agalactiae. In the invention there is developed recombinant plasmid DNA pQE-pB1 representing plasmid DNA pQE-30 that bears recombinant DNA pB1, and strain-producer E. coli M15-PB1 enabling to express recombinant protein PB1.

EFFECT: no enzymatic activity of produced recombinant protein allows application as an ingredient of the vaccine for Streptococcus pyogenes and Streptococcus agalactiae.

7 cl, 7 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to genetic engineering and can be used in medicine. The mucosal vaccine contains effective amount of hybrid protein consisting of oncoprotein E7 of human papilloma virus fused with heat-shock protein of mycobacteria Hsp70, chitosan related to hybrid protein 1:0.1-10 and additives pharmaceutically acceptable manufacturing of suppositories. The mucosal vaccine is used in therapy of the diseases associated with human papilloma virus.

EFFECT: possibility for multiple improvement of clinical effectiveness of diseases associated with human papilloma virus, considerable reduction of treatment cost in comparison with common techniques of treating cervical carcinoma and "РПК"; elimination of injection by-effects undesirable and extremely dangerous for the patent's life, eg anaphylactic shock, owing to local application; simplification of medical process - the patient can receive medical treatment out of clinic by independent introduction of the preparation.

6 cl, 4 dwg, 2 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: invention concerns protein NMB1870 which represents common surface protein Neisseria meningitidis expressed with all Neisseria serogroups. Protein is subdivided into three separate families. The whey induced against antigen of a certain family, has bactericidal effect within this family, but is inactive concerning strains expressing antigens of one of other two families, i.e. there is a cross protection within family, but not between families. It is established, that NMB1870 can be subdivided into domains and that antigen domains can be recovered from NMB1870 of all three families and expressed as a polypeptide chain that is implemented by the method disclosed in the invention. Also it is discovered that NMB1870 expresses some epitopes in surface loops located between alpha spirals, and that epitope substitution of the loop of one family with that of the other family enables making chimeric NMB1870 of antigenicity characteristic for proteins of several families. In the invention there are disclosed chimeric proteins NMB1870 (versions) partially containing NMB1870 of various families.

EFFECT: proteins produced according to the invention can be used as a medical agent as a component of immunological compositions for immunization against diseases caused by Neisseria meningitidis without family specificity of protection.

22 cl, 46 dwg

FIELD: medicine; microbiology.

SUBSTANCE: way is intended for reception of functionally active LF form, the basic toxic protein defining cellular disturbances, leading to death of an organism at infection with a malignant anthrax bacterium. For realisation of the way a recombinant plasmid pETHIS-LF (7816 items) is designed, containing a full-size gene of the lethal factor (LF) of malignant anthrax under the control of the promotor of bacteriophage T7 and to a determinant of ampicillin tolerance. The plasmide provides effective synthesis of LF protein of malignant anthrax merged with sequence of six Histidinums for clearing with the metal-chelate chromatography. The strain Escherichia coli BL-HISLF is designed using transformation of the specified plasmid DNA in the strain E.coli BL21 (DE3), synthesizing active LF protein. The target product is separated with the way including clearing on a metal-chelate sorbent with the subsequent additional clearing of the LF protein by gel-filtration.

EFFECT: reception of active recombinant protein LF on the simplified technology and with a high output of synthesised protein of the lethal factor.

3 cl, 3 dwg, 3 ex

FIELD: biology.

SUBSTANCE: designed is a new recombinant plasmid pETGST-LFmin (7704 nucleotide pairs), containing a catalytically active fragment of the gene of lethal factor anthrax (LF) controlled by a bacteriophage T7 promoter, determinant of resistance to ampicillin and a glutathione-S-transferase sequence for efficient purification of the recombinant protein on a sorbent with immobilised glutathione. The plasmid provided for efficient sythensis of the protein of LF anthrax, chimerised with a sequence of glutathione-S-transferase for purification on immobilised glutathione. Escherichia coli BL-LFminGST strain is obtained from transformation of the said plasmid DNA into a E.coli BL21 (DE3) strain, which provides for output of synthesised LF protein of not less than 90 mg/l g of raw biomass. The active LF protein is obtained using a method which involves culturing the said recombinant strain, destruction of bacterial cells in a buffer solution with pH 7.4 in the presence of Triton X-100 and a protease inhibitor, and purification on a sorbent with immobilised glutathione.

EFFECT: output of proteolytic active recombinant chimeric purified protein of LF anthrax in amount of not less than 70 mg/l g of raw biomass.

3 cl, 3 dwg, 3 ex

Neisseria antigens // 2347813

FIELD: chemistry; biochemistry.

SUBSTANCE: invented here are proteins of the meningococcus bacteria Neisseria meningitides (mainly strain B), with immunogenic properties. The proteins have defined amino acid sequences, presented in the description, and are coded with corresponding nucleotide sequences. Description is also given of an antibody, specific to the indicated meningococcus proteins. These proteins, coding their nucleotide sequences, as well as the specific antibody, can be used as an active ingredient in compositions for treating or preventing infection caused by Neisseria meningitides. The presented proteins can be used as antigens for making effective vaccines, immunogenic compositions.

EFFECT: obtaining proteins, used as ingredients for making effective vaccines, immunogenic compositions.

11 cl, 2 tbl, 104 ex

FIELD: medicine; biotechnologies.

SUBSTANCE: amino-acid sequences are presented in the list of the sequences obtained from a bacterium, mainly strains A and B which show properties of an antigene. The invention includes corresponding nucleotide sequences of fragments of the nucleinic acid, coding amino-acid sequences of the specified proteins. The proteins under the invention can be used as antigenes for obtaining of specific antibodies and manufacturing of compositions for treatment, preventive maintenance or diagnostics of the infection caused Neisseria meningitidis. The compositions are prepared on the basis of a protein, a fragment of nucleinic acid or an antibody with addition of the pharmaceutically comprehensible carrier and represent vaccinal, diagnostic or pharmaceutical compositions. Sequences of the obtained proteins have no any considerable homology with sequences Neisseria gonorrhoeae, that allows to receive treatment-and-prophylactic and diagnostic compositions with high specificity in relation to N. meningitidis and also to receive Diagnostic reagents for differentiation N. meningitidis from N. gonorrhoeae.

EFFECT: efficiency of application.

12 cl, 2 tbl, 17 ex

FIELD: medicine, microbiology.

SUBSTANCE: invention concerns nucleotide sequence coding TolC, and also to certain amino-acid sequence which is built in in permissive, located from lateral aspect of a membrane area of TolC. In the invention is described a plasmid, containing a nucleotide sequence, for an expression of the merged protein or merged peptide. The invention concerns also the transformed bacterium, in particular enterobacteria, to its use as a part of a pharmaceutical composition for stimulation of the immune response and in a diagnostic set for detection of interesting substance in an organism. The framed transport system provides high efficiency of presentation of a product of an expression in an external cellular membrane of a bacterium.

EFFECT: provision of high efficiency of presentation of product of expression in external cellular membrane of a bacterium.

13 cl, 1 dwg, 5 ex

FIELD: medicine; pharmacology.

SUBSTANCE: polypeptides under the invention include amino-acid sequence, structurally related sequence SEQ ID NO:1 resulted in the text of the description, to use of such polypeptides as immunogens, including as a part of compositions. The invention opens also expression systems for producing such polypeptides. The sequence SEQ ID NO:1 represents a truncated derivative of full-size polypeptide S. aureus. This full-size polypeptide is designated in the invention as full-size "ORF0657n". It is established, that the polypeptides described in the invention and containing amino-acid sequence are SEQ ID NO:1.

EFFECT: polypeptides cause protective immune response against staphylococcus aureus.

43 cl, 56 dwg, 4 tbl, 17 ex

FIELD: chemistry.

SUBSTANCE: invention relates to polypeptides with established amino acid sequence, which have antimicrobial activity. Amino acid polypeptide sequence has 65-99% of identity with amino acid sequence of polypeptide, originating, particularly, from strain Pszudoplectania nigrella CBS 444.97. Invention also relates to polynucleotide, which has nucleotide sequence, coding said polypeptide, constructions of nucleic acid, recombinant expression vector, which contains said construction, and recombinant host-cell, intended for obtaining said polypeptide. In invention methods of obtaining polypeptide and inhibiting microbe cell growth using said polypeptide are described. According to invention polypeptides can be used for production of veterinary or therapeutic medication for treatment or prevention of microbial infection in humans and animals. According to invention polypeptide with antimicrobial properties ensures stability of antimicrobial activity when used in different forms of antimicrobial medications.

EFFECT: obtaining polypeptides with antimicrobial properties.

19 cl, 3 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: there is designed recombinant plasmid DNA pSC13D6 containing a gene of one-chained antibody to tick-borne encephalitis virus sc13D6. Plasmid consists of 3782 basepairs and contains: a plasmid vector pGEMl, phage T7 promotor and unique restriction sites. There is also discovered strain of bacteria Escherichia coli BL21 (DE3)/pSC13D6 - a producer of a virus neutralising one-chained antibody sc13D6 to tick-borne encephalitis virus.

EFFECT: invention allows producing one-chained antibodies sc13D6 to tick-borne encephalitis virus able to inhibit it effectively.

2 cl, 5 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: invention can be used in manufacturing of vaccines for Streptococcus pyogenes - streptococci of group A (SGA) and Streptococcus agalactiae - streptococci of group B (SGB). Substance of the invention involves development of recombinant DNA pB1 derived from PCR with using chromosomal DNA of strain 090R Ia of serotype SGB, primers Pb1 and Pb2 and following cloning with using expression plasmid pQE-30 in E coli M15. Recombinant DNA pB1 codes recombinant protein PB1 expressing protective properties in relation to specified streptococci which has no enzymatic activity and causes synthesis of anti-Pb1 antibodies expressing protective properties in relation to Streptococcus pyogenes and Streptococcus agalactiae. In the invention there is developed recombinant plasmid DNA pQE-pB1 representing plasmid DNA pQE-30 that bears recombinant DNA pB1, and strain-producer E. coli M15-PB1 enabling to express recombinant protein PB1.

EFFECT: no enzymatic activity of produced recombinant protein allows application as an ingredient of the vaccine for Streptococcus pyogenes and Streptococcus agalactiae.

7 cl, 7 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: there are described recombinant plasmids pL1spCBD and pL2spCBD. There are offered strains Escherichia coli that are producers of chimeric proteins L1spCBD and L2spCBD. There are described recombinant proteins L1spCBD and L2spCBD. There is disclosed method of immobilisation, concentration and purification of proteins L1spCBD and L2spCBD both cellulose and polystyrene carriers.

EFFECT: specific interaction of recombinant proteins L1spCBD and L2spCBD with specific blood serum antibodies recovered from sick and vaccinated patients and the absence of interaction with serums recovered from healthy people and immunised patients.

8 cl, 1 dwg, 6 ex

FIELD: biotechnologies.

SUBSTANCE: invention is related to the field of biotechnology, specifically, to separation and identification of new genes of spiramycins biosynthesis track and to new polypeptides, which participate in this biosynthesis, and may be used to produce acyltransferase, which is responsible for modification of platenolid in position 3. Polynucleotide coding acyltransferase, which is responsible for modification of platenolid in position 3, cells of bacterium Streptomyces type are transformed, and strain-producer of end polypeptide is made.

EFFECT: increased extent of production and purity of produced spiramycin.

26 cl, 41 dwg, 44 tbl, 31 ex

FIELD: biotechnologies.

SUBSTANCE: invention is related to biotechnology and may be used for site-specific hydrolysis of DNA, which contains C5-methylcytosin bases. Strain Paracoccus carotinifaciens 3K is extracted from soil, which provides for production of site-specific endonuclease, which recognises and splits both chains of nucleotide sequence of DNA containing four C5-methylcytosin bases in site of recognition 5'-WCGNNNNNNNCGW-3', with production of single-nucleotide 3'-protruding ends.

EFFECT: invention makes it possible to produce new site-specific endonuclease Pcsl, which may be used for detection and analysis of methylated DNA.

2 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: invention is related to mutant of rumen bacterium of MANNHEIMIA type, which is produced by inactivation of lactate dehydrogenase gene (ldhA) and pyruvate-formate lyase gene (pfl). Mutant of rumen bacteria of MANNHEIMIA type, which is produced by inactivation of lactate dehydrogenase gene (ldhA), pyruvate-formate lyase gene (pfl), phosphotrans acetylase gene (pta), and acetate kinase gene (ackA). Mutant of rumen bacteria of MANNHEIMIA type, produced by means of inactivation of lactate dehydrogenase gene (ldhA), gene of pyruvate-formate lyase (pfl) and gene of phosphoenolpyruvate carboxylase (ppc). To methods for production of succinic acid, by cultivation of above mentioned mutants in anaerobic conditions.

EFFECT: bacterial mutants of the present invention are able to produce succinic acid in high concentration, while production of other organic acids is reduced or practically unavailable.

28 cl, 13 dwg, 1 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: recombinant strain Escherichia coli is produced, which contains plasmid pHINS21 (Escherichia coli JM109/ pHINS21), defining synthesis of hybrid protein, made of N-terminal fragment of human gamma-interferon and human proinsulin, joined by peptide linker, which contains site of splitting with enterokinase (Asp4Lys). Yield of hybrid product that includes proinsulin, provided with new strain-producer, makes at least 30% of total amount of cell protein. Method is suggested for preparation of human proinsulin, including cultivation of strain-producer Escherichia coli JM109/pHINS21, separation of inclusion bodies and their dissolution, renaturation of hybrid protein and its cleaning with ion-exchange chromatography, splitting of hybrid protein with enterokinase or its catalytic subunit and cleaning of proinsulin by ion-exchange chromatography on sorbates with sulfprofile groups.

EFFECT: invention simplifies technological process for production of recombinant human proinsulin and improves conditions of its execution from the point of view of safety engineering.

4 cl, 4 dwg, 5 ex

FIELD: food industry.

SUBSTANCE: strain Streptococcus thermophilus which produces lactic acid is described. Sequence of nucleic acids made of the strain producing polysaccharides are also described as well as food or pharmaceutical composition and milk product containing such strain.

EFFECT: strain has strong structural properties.

16 cl, 4 dwg, 6 tbl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: invention is related to biotechnology, in particular to production of n-butanol by means of carbohydrate-containing raw material fermentation with recombinant bacteria. The following plasmid DNA are constructed: pBCS, containing genes of butanol biosynthesis crt, bed, etfB, etfA and hbd from C.acetobutylicum and pTHL-BCS, containing, apart from earlier mentioned, gene thi from C.acetobutylicum. Plasmids may be replicated both in gram-negative (E.coli) and in gram-positive (L.brevis) bacteria. Recombinant strain-producers of n-butanol are produced on the basis of bacteria Lactobacillus brevis: strain Lactobacillus brevis VKPM V-10044, containing plasmid pBCS; strain Lactobacillus brevis VKPM V-10043, containing plasmid pTHL-BCS, which are able to synthesise butanol and resistant to its concentration of 2.0-2.8 wt % in liquid medium. Method is developed for synthesis of butanol on the basis of recombinant bacteria L.brevis, which combine ability to synthesise butanol with resistance to its concentrations in liquid medium of at least 2.0 wt %, which makes it possible to produce butanol using both glucose and xylose as source of carbon in mediums for cultivation.

EFFECT: invention makes it possible to increase synthesis efficiency.

5 cl, 2 tbl, 8 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and is a method of producing staphylokinase using an OXY-1 cassette with sequence SEQ ID No. 1. The cassette is part of two plasmids with international inventory numbers BPL-0020 and BPL-0021, which are transformed strains of bacteria E. coli for producing staphylokinase.

EFFECT: possibility of obtaining highly effective staphylokinase.

16 cl, 11 dwg, 4 ex

FIELD: pharmacology.

SUBSTANCE: present invention refers to immunology and biotechnology. There is offered recovered human antibody to RG1 polypeptide. There are described versions of antibodies, including one-chain antibody, and immunoconjugate based on said antibodies. There are disclosed methods of selective cell destruction, cell inhibition, treatment of disease state, detection of disease state, detection of RG1, monitoring of clinical course of prostate cancer, prediction in a person with using antibodies and immunoconjugate.

EFFECT: application of the invention provides new antibodies to RG1 polypeptide that can find application in treating tumours with RG1 overexpression.

16 cl, 4 dwg, 1 tbl, 13 ex

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