Method of differentiation of strains of principal and nonprincipal causative plague agent and pseudotuberculosis agent by polymerase chain reaction
SUBSTANCE: method provides recovery of DNA of an analysed strain followed by PCR with using nucleotide primers of terC, ilvN and inv genes of the following sequences: 89-S - AATCAAATCTCGCCCAGC, 89-As -GCTGCGTATCATTTCACC; 45-S - AGTGGTCTGCTTCTCTGG, 45-As -CGGCATACACAGAATACC; inv839 - TACCTGCACTCCCACAAC, inv1007 -CCCATACGCTGATCTACC. The analysed strains are differentiated by matching the sizes of the derived fragments of terC, ilvN and inv genes with the similar fragments in typical strains of principal and nonprincipal plague agents.
EFFECT: invention allows quick, effective and reliable differentiation of the strains.
1 tbl, 3 ex
The invention relates to the field of medical Microbiology, in particular to subspecific differentiation of strains of the plague pathogen and differentiation of plague and pseudotuberculosis microbe.
Plague pathogen which is a type of bacteria, Yersinia pestis, still poses a real threat to human health associated with existence on different continents numerous active natural foci of plague, and the use of the plague pathogen in bioterroristic acts. Every year in the world there are a sufficiently large number of cases of plague, which often lead to death. Because of this importance is the development of accurate methods of detection Y.pestis and differentiation of this agent from other bacteria, in particular from the closely related Yersinia pseudotuberculosis. The look of Y.pestis heterogeneous in composition and is divided into the main subspecies subspecies (ssp) pestis and group non-core subspecies: Altai - ssp.altaica, Caucasian - ssp.caucasica, wagasky - ssp.ulegeica, Hissar - ssp.hissarica. The subspecies of the plague pathogen differ in virulence and epidemiological significance. The strains of the main subspecies are characterized by high virulence and high epidemic potential. Strains of non-core races have selective virulence, epidemically insignificant and hardly cause for which olivani person. In this regard, along with the need for differentiation of plague and pseudotuberculosis microbe is not less important and differentiation of subspecies Y.pestis, and, in particular, the detection of these strains of the main subspecies. The development of efficient and reliable methods for rapid identification of the main subspecies of Y.pestis among closely related pathogenic Yersinia has important practical value.
Currently, the differentiation of strains of the plague pathogen main and non-main subspecies carried out taking into account the different expression of a number of biochemical characteristics: the fermentation of monosaccharides - ramnose, melibiose, products isocitrate and others. However, the phenotypic manifestation of the plague pathogen properties depends on the conditions of cultivation of strains, the quality of the environments, and the isolation of the pathogen in pure form, which greatly complicates and increases the time of analysis.
Greater efficiency and speed of obtaining results different method of polymerase chain reaction (PCR), based on the use of genetic characteristics of strains of various bacteria. For PCR does not necessarily isolation of pure cultures of the pathogen, since the method is highly sensitive and allows the determination of species and subspecies belonging to b is Cherie if you have a small number of cells in the material of the test sample.
Up to the present time PCR was used for detection of the causative agent of plague, as well as to differentiate it from the causative agent of pseudotuberculosis, but almost never used it to differentiate between main and non-main subspecies of Y.pestis. For detection of the causative agent of plague in PCR used the nucleotide sequences of various genes caf, pla, lcrV, 3a, irp-2 (Neubauer H. et al., 2000; Rahalison L. et al., 2000; Hongwei Z.J.L., 2000; Balakhonov S.V. with coawt; Garanina S.B. et al., 2001; Knots YU et al., 2001; L. Radnedge et al., 2001; Savostin, H.E., 2004; Kuske C.R. et al., 2006; Kuklev B.E. et al. 2006).
So, there is a method of detecting bacteria of the genus Yersinia and differentiation of a human pathogenic species of Yersinia by polymerase chain reaction (patent RU 2354700 published 10.05.2009, on the basis of sequence portion of a gene ompF Porin. This method allows you to split the pathogenic species of Yersinia - causative agents of plague and pseudotuberculosis, intestinal yersiniosis at the species level, but it does not allow for identification of Y.pestis strains of main and non-main subspecies.
Known another method for rapid detection and differentiation of Y.pestis and Y.pseudotuberculosis (DE 10124342 (A1), published 28.11.2002 g), which provides a two-step detection of the DNA of the causative agents of plague and pseudotuberculosis: PCR and hybridization with specific probes, which increases n is dejnost and efficient way. However, this method also does not allow to differentiate strains of Y.pestis main and non-main subspecies.
Closest to the proposed invention, the essential features is the way of differentiation of the plague and pseudotuberculosis microbe with simultaneous intraspecific differentiation of strains of the plague microbe (EN 2332464 published, 27.08.2008), which is based on the use of the variability of the nucleotide sequence of a segment of a chromosome hutG - YP01967. However, the disadvantage of this method is that the plot hutG - YP01967 is located in the unstable region of the genome Y.pestis, which can often be lost in strains of the main subspecies, which makes it impossible for the differentiation of these strains using the proposed method. In addition, this method is difficult to interpret, requires the use of a large group of reference strains, and also has some error, because it does not allow to avoid coincidence of the sizes of PCR fragments from some strains of the main and non-main subspecies, which requires additional research (e.g., sequencing) to determine the subspecific affiliation of strain. Other publications about the use of the PCR method to differentiate strains of the plague pathogen main and non-main subspecies in the literature are absent, as are absent, and given what's about the parts of the genome Y.pestis, the use of which would allow to quickly and efficiently perform such differentiation.
The objective of the invention is to develop a simple interpretation, reliable and rapid method for the differentiation of strains of the plague pathogen main and non-main subspecies and strains of the causative agent of pseudotuberculosis by PCR method.
The technical result consists in increasing the efficiency of differentiation of strains of the plague pathogen main and non-main subspecies with simultaneous differentiation of strains of the causative agent of pseudotuberculosis.
The inventive method is based on using as a DNA target of chromosomal genes livelihoods terC, ilvN and inv, encoding respectively the protein resistance tellurium, acetolactate and invasin-adhesin.
The technical result is achieved by the method of differentiation of strains of the plague pathogen main and non-main subspecies and the causative agent of pseudotuberculosis by polymerase chain reaction, which involves the isolation of the DNA of strain, carrying out polymerase chain reaction with amplification of the gene fragments terC, ilvN and inv investigated strain, while the primers for these genes have the following sequence:
89-S - AATCAAATCTCGCCCAGC,
89-As - GCTGCGTATCATTTCACC;
45-S - AGTGGTCTGCTTCTCTGG,
45-As - CGGCATACACAGAATACC,
inv839 - TACCTGCACTCCCACAAC,
inv1007 - CCCATACGCTGATCTACC,
and dimensioning am limitirovany fragments with subsequent differentiation of the studied strains, which is carried out by comparing the sizes of the obtained gene fragments terC, ilvN and inv with similar fragments in the typical strains of the plague of the main and non-main subspecies.
The method is as follows.
The isolation of the DNA of the studied strains of the plague microbe performed by standard methods in accordance with MU 1.3.1794-03 "Organization of work in the research by PCR material infected by microorganisms 1-11 of pathogenicity groups.
Polymerase chain reaction carried out according to standard methods (MU 1.3.1794-03) using the above primers for genes terC, ilvN and inv. Oligonucleotide primers used for amplification in PCR fragments terC, ilvN and inv, calculated on the basis of the nucleotide sequences of these genes in strains of the plague microbe Y. pestis CO92, KIM, Antiqua, Nepal516, Angola, 91001, Pestoides F and Y. pseudotuberculosis IP31758, presented in the database of NCBI GenBank. Using designed primers is carried out in PCR amplification of gene fragments terC, ilvN and inv and determines the size of the obtained gene fragments. Primers for the genes terC, ilvN and inv have the following sequence:
89-S - AATCAAATCTCGCCCAGC,
89-As - GCTGCGTATCATTTCACC;
45-S - AGTGGTCTGCTTCTCTGG,
45-As - CGGCATACACAGAATACC,
mv839 - TACCTGCACTCCCACAAC,
invl007 - CCCATACGCTGATCTACC.
Polymerase chain reaction with amplification of the gene fragments terC, ilvN and inv carry out the following program: cycle of 94°C for 5 min, then 35 cycles at 94°C 45 sec, 55°C 1 min, 72°C 45 s and a final cycle of 3 min at 72°C. the sizing educated in PCR fragments of genes terC, ilvN and inv in the tested strains carried out by electrophoresis in 3% agarose gel relative to the standard markers molecular masses ranging in size from 100 to 1000 BP We found that the typical strains of the plague pathogen main subspecies are formed in PCR fragments of genes terC - 300 gel, ilvN - 515 P.N. and inv - 877 P.N.; minor strains of the subspecies accordingly, 389, 560 and 877; and the strains of Yersinia pseudotuberculosis microbe - 389, 560 and 169 (table). Further differentiation of the studied strain is carried out by comparison with the data presented in the table.
The invention is illustrated by examples.
Example 1. Differentiation of strain No. 1 (Model experiment).
The isolation of the DNA of strain No. 1 performed by a standard method using a lytic solution based on 6M guanidinosuccinic preliminary disinfection of culture by adding thimerosal sodium concentrations up to 1:10000, followed by heating at a temperature of 56°C for 30 minutes (the Organization of work in research by PCR material infected with microorganisms of I-II pathogenicity groups. MU 1.3.1794-03).
Polymerase chain reaction is carried out in a volume of 25 μl; the reaction mixture contains: 1x buffer (10x Ptrbuffer - 2,5 mm), MgCl22.0 mm dNTP mixture - 0,3 mm oligonucleotide primers - 2 pmol, Taq DNA polymerase, and 0.1 units studied DNA 10 ál. Amplification products analyzed in 3% agarose gel with the addition of ethidium bromide and viewed under UV light.
Determine the sizes of the resulting fragments terC genes, ilvN and inv. They are 300, 515, 877 BP, which corresponds to the size of fragments of genes in strains of the main subspecies. The investigated strain No. 1 referred to the main subspecies of Y.pestis.
Example 2. Differentiation of strain No. 2 carried out analogously to example No. 1.
Dimensions formed in PCR fragments of genes terC, ilvN and inv amount 389, 560 and 877 BP, which corresponds to the sizes of the fragments produced in strains of the plague pathogen in non-core races. Strain No. 2 referred to as non-core subspecies of Y.pestis.
Example 3. Differentiation of strain No. 3 carried out analogously to example No. 1.
Dimensions formed in PCR fragments of genes terC, ilvN and inv amount 389, 560 and 169 BP, which corresponds to the sizes of the fragments produced in strains of the causative agent of pseudotuberculosis. Strain No. 3 referred to Y.pseudotuberculosis.
Thus, the claimed method of differentiation of strains of the plague pathogen main and non-main subspecies PCR, based on the variability of gene sequences livelihoods terC, ilvN and inv allows you to quickly, efficiently and reliably carry out the differentiation of States the MOU Y.pestis main and non-main subspecies and Y.pseudotuberculosis.
|Species, subspecies||Dimensions genes|
|Y.pestis main subspecies||300||515||877|
|Y.pestis non-main subspecies||389||560||877|
The method of differentiation of strains of the plague pathogen main and non-main subspecies and the causative agent of pseudotuberculosis, including DNA isolation, PCR with amplification of gene fragments of the studied strain, characterized in that when carrying out PCR using nucleotide primers for the genes terC, ilvN and inv with the following sequence:
89-S - AATCAAATCTCGCCCAGC,
89-As - GCTGCGTATCATTTCACC;
45-S - AGTGGTCTGCTTCTCTGG,
45-As - CGGCATACACAGAATACC;
inv839 - TACCTGCACTCCCACAAC,
invl007 - CCCATACGCTGATCTACC;
differentiation of strains is carried out by comparison of the sizes of the obtained gene fragments terC, ilvN and inv issleduemymi with similar fragments in the typical strains of plague primary, non-main subspecies and Yersinia pseudotuberculosis microbe, with typical strains of the plague pathogen main subspecies are formed in PCR fragments of genes terC - 300 gel, ilvN - 515 P.N. and inv - 877 P.N.; minor strains of the subspecies, respectively, 389, 560 and 877; and the strains of Yersinia pseudotuberculosis microbe - 389, 560 and 169.
SUBSTANCE: what is offered is a method of marking a human 7th chromosome providing in situ hybridisation of metaphasic or interphasic cell chromosomes of a tested sample and a DNA probe presented by a marker plasmid alpha R1-13 which consists of EcoR 1-EcoR l DNA fragment of a pBR 325 vector of the value 5966 base pairs and EcoRl-EcoRl alphoid DNA fragment of the human 7th chromosome of the value 680 base pairs. The testing environment in which the used DNA-probe specifically interacts with a centromeric region of the 7th chromosome without cross hybridisation with other human chromosomes are developed.
EFFECT: more efficient identification of the presented chromosome and enabled application of the new method in medical diagnostics.
SUBSTANCE: reactions are conducted in the same PCR-microtube on walls of which there is a probe immobilised. In comparison with standard, the PCR-tubes used have a feature of construction: an extended internal surface, and as consequence - a great sorption capacity. A process of selective amplificate sorption on the test tube walls occurs after each anneal stage: temperature decreasing leads to hybridisation of amplicon chain not only among themselves, and with probes preliminary immobilised on the test tube walls. Using the similar PCR-microtubes enables increasing sensitivity of an immune-enzyme assay of amplicons and decreasing a number of polymerase chain reaction cycles. The fixed analytical signal will characterise both the presence of the required DNA, and show its concentration in the sample.
EFFECT: offered test system can be adapted for any existing PCR-technique, does not require special instrumentation and is reliable, simple and efficient for all parameters.
6 cl, 1 dwg, 1 tbl, 1 ex
SUBSTANCE: during selection by markers, a set of alleles related to loci of quantity tokens (QTL) that contribute to expression of certain phenes of economic value is introduced into a corn idioplasm. The criteria are selected among grain crop capacity, grain moisture when picked, early and late root fit, stem drowning, frequency of common rust, frequency of ear rotting caused by Fusarium (ear wilt), resistance to Sulcotrione and panicle structure. Invention also relates to the method of such plants production, and also to methods of analysis and screening to identify plants with a required profile of alleles.
EFFECT: improved method of new corn plants production.
33 cl, 1 dwg, 19 tbl
SUBSTANCE: synthetic oligonucleotides for indentifying DNA of Torque teno virus of all known genotypes are disclosed. Primers are combined in a set for DNA identification in blood and other biomaterials of the infectious agent of the latent viral infection Torque teno virus of Circoviridae family by polymerase chain reaction.
EFFECT: invention allows reliable identification of said virus in a biological material.
SUBSTANCE: analysed sample is studied simultaneously by two methods: the first one is a indirect immunofluorescence (IIMF) reaction, while the second one involves a polymerase chain reaction (PCR); the IIMF provides using monoclonal antibodies "BCKK" (P-384D and 434D). The antibodies interact with the capsular antigen F1 specific for Y.pestis species, or the plasmid-temperature-independent surface protein PFV which is found in all Y.pestis strains and rare R-form Y.pseudotuberculosis strains. The bacteria luminescence shows the presence of native or fraction-less Y.pestis bacteria, or typical and PFV-atypical Y.pseudotuberculosis strains in the sample. The PCR is conducted by two pairs of primers vlm33for/ISrevl754 - specific for Y.pestis species, and JS - specific for Y. pseudotuberculosis species. The values derived with the first pair of the primers are estimated as positive if observing the amplicons in 400 bps, and with the second pair if observing the amplicons in 223 bps; the analysed sample is identified by the matching the IIMF and PCR values with the reference strains.
EFFECT: method provides quick identification and high-reliability differentiation of all strains.
7 tbl, 6 ex
SUBSTANCE: method for bioindication of water bodies involves collecting samples of planktons inhabiting in a water body, determining the contamination level by analysing said samples and assessing the analysis results. The contamination level is determined via phylogenetic analysis of ribosomal RNA genes (18S rRNA) of planktons in the sample. Phylogenetic trees built from the conservative 18S rRNA gene are determined and evolutionary relationships of the analysed object with other saprobionts are identified. Analysis results are assessed as follows: at high (over 85%) value of bootstrap support of clusters containing the analysed planktons and resistant saprobionts, the following conclusions are made: resistant indicator organisms xeno- or oligosaprobic (or exclusively xenosaprobic) of water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in a safe ecological state and there is no threat of negative anthropogenic action, if resistant indicator organisms oligo- and mesosaprobic (or exclusively oligosaprobic) of the water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in an unstable (transition from safe to unsafe state) ecological state, is under insignificant anthropologic load, is capable of self-recovery and does not need additional environmental protection measures, if resistant indicator organisms meso- and polysaprobic (or exclusively mesosaprobic) of water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in an unsafe state and is under considerable anthropologic load, natural capability of self-recovery is insufficient and the water body needs environmental protection measures, if resistant indicator organisms of polysaprobic water bodies and the analysed plankton merge into one cluster, it is concluded that there is a local ecological disaster and there is need for urgent recovery measures.
EFFECT: high reliability of the biomonitoring result for use without territorial limit, independent of the geographical location of the investigated water body.
SUBSTANCE: bacteria freely immobilised on a slide are processed with a lysis solution containing an ionogenic protein-denaturing detergent and EDTA. Further, DNA-nucleoid is dry heating and microwave incubation stabilised. DNA is stained by high-sensitivity DNA-specific fluorochrome, in conclusion DNA integrity is evaluated. Offered is a kit for evaluating the DNA bacteria integrity which comprises all the components required.
EFFECT: invention can be used for determining the bacterial DNA fragmentation levels by a precise express technique.
16 cl, 11 dwg, 5 tbl, 9 ex
SUBSTANCE: offered invention can be used for the quantitative determination of alive bacteria. The offered method involves PCR-based product amplification with using the primers able to specific hybridisation with rRNA of a bacterium of interest. It is followed with the analysis of cell number of the alive bacterium of interest taking into account PCR cycle number.
EFFECT: invention allows precise determination of cell number of the alive bacterium of interest in a test sample.
8 cl, 7 dwg, 4 tbl, 10 ex
SUBSTANCE: it is described that immunogenicity of hemagglutinin (HA) molecule of influenza virus can be increased by substituting amino acids in the HA sequence. Substituting specific residues in HA such as asparagine introduction in position 223 in HA H5 allows ensuring more sensitive hemagglutination inhibition (HI) test provided by changing receptor specificity and/or ability to antibody-antigen linkage. The HA molecules having such substitutions can find application in creating diagnostic prototype viruses.
EFFECT: improved influenza virus vaccines.
9 cl, 3 dwg, 7 tbl, 7 ex
SUBSTANCE: specified plant is a plant of Cucumis sativus type and includes at least one area at one chromosome, which gives resistance to closterovirus, and at least one area, which gives resistance to powdery mildew. The area of the chromosome, which gives resistance to closterovirus, is linked to at least one marker selected from the group, made of markers E16/M50-244, E16/M50-188, and E11/M48-251. The area of the chromosome, which gives resistance to powdery mildew, is linked to at least one marker selected from the group made of the following components: a marker of single-nucleotide polymorphism 39T→G in SEQ ID NO:1, a marker of single-nucleotide polymorphism 29G→A in SEQ ID NO:2, a marker of single-nucleotide polymorphism 193C→T in SEQ ID NO:3, mutation of an insert 5'-AATTT-3' in position 221 in SEQ ID NO:4, and markers E16/M50-F-194, E11/M48-F-251, E23/M38-M001, E23/M40-M003, E24/M46-M002, E24/M46-M003, E12/M91-M003, E26/M43-M003, E14/M59-F-134 and E14/M59-F-200.
EFFECT: production of the plant resistant to closterovirus and to powdery mildew of cucumbers.
25 cl, 8 dwg, 4 tbl
SUBSTANCE: invention describes mutations of the amino acid sequence of the protein of atpE mycobacteria (M. tuberculosis and M. smegmatis), which determine their resistance to DARQ J. Obtained are coding mutant forms of atpE nucleic acids, vectors containing said forms and host cells expressing mutant proteins.
EFFECT: invention discloses use of said host cells in a method of identifying compounds which can be used as antimicrobial agents for mycobacteria strains resistant to existing drugs.
5 cl, 4 dwg, 7 tbl
SUBSTANCE: polypeptides exhibit antimicrobial activity and recovered polynucleotides coding these polypeptides. Nucleic acid constructs, vectors and host cells contain said polynucleotides.
EFFECT: applicability of polypeptides in veterinary science and medicine, as well as in forage production.
16 cl, 6 tbl, 7 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention concerns biotechnology and health care. Recombinant adenovirus p53 is capable of lowering side effects of the type caused by anti-tumour chemotherapy and radio-therapy. In cancer patients, recombinant adenovirus p53 can restore operation of organs, including blood analysis, liver functioning, kidney functioning etc in order to improve the quality of life of patients, for example improvement of appetite, mental status and the like.
EFFECT: invention can be used in medicine.
19 cl, 7 dwg, 4 tbl, 5 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: compounds, included into one of compositions represent polypeptides, which contain at least immunogenic fragment of protein CT858 or protein CT089 Chlamydia trachomatis with specific amino acid sequence, or polynucleotides, coding immunogenic fragments of said proteins. Compounds, included into other composition, represent fused polypeptides, which contain immunogenic fragment of protein CT858 or CT089 and fusion partner, selected from immunologic fusion partners, expression amplifiers, affine markers and unrelated known proteins of Chlamydia trachomatis. Said compositions can be used for stimulation of specific T-cell response to Chlamydia trachomatis, for inhibition of infection development and for treatment of infection induced by Chlamydia trachomatis, ensuring high level of immune response and therapeutic effect.
EFFECT: described are compositions based on compounds for prevention and treatment of Chlamydia infection.
11 cl, 16 dwg, 7 tbl, 13 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology and is a lipolytic enzyme which is obtained from one Streptomyces. Such a lipolytic enzyme has an amino acid sequence reduced to SEQ ID No.4, or an amino acid sequence which is at least 70% identical to it. The invention also relates to use of a lipolytic enzyme in a method of producing lyso-glycolipid, lysophospholipid, in an oil refining method, in phospholipid processing methods, in bio-conversion of polar lipids and in production of food products.
EFFECT: invention enables to obtain a novel enzyme having glycolipid hydrolysing action.
46 cl, 17 dwg, 4 tbl, 12 ex
SUBSTANCE: polypeptide homological to polypeptide expressed by Eurotinium amstelodami, has antimicrobial activity and may be used to administer as medical or prophylactic agent to a person or animal, and also in fodder for animals.
EFFECT: improved efficiency of application.
19 cl, 2 tbl, 6 ex
SUBSTANCE: invention can be used in producing vaccines against Streptococcus agalactiae - a representative of streptococci group B (SGB) in diagnostics of the diseases - for creation of a detection system of immunoglobulin A level in biological fluids, in immunochemistry as accessible immunochemical reagents (affine recovered IgA fragments). Offered unique recombinant DNA are produced by polymerase chain reaction (PCR) with using chromosomal DNA of strain 219/4849 Ibc of serotype SGB and unique primers. One recombinant DNA contains three nucleotide substitutes in comparison with an initial site of chromosomal DNA. The following cloning of amplified fragments is carried out in a linear vector pGEM-T Easy, and at the final stage by the system of express ionic vectors pQE30/31/32 in E coli JM 109. The produced recombinant DNA code amino acid sequences of recombinant polypeptides exhibiting ability to connect selectively various molecular forms of IgA and designated as P6, P7, P8. Polypeptide P6 causes synthesis of long circulating high-affine anti-Rb antibodies possessing protective properties against SGB.
EFFECT: application of the invention provides production of recombinant polypeptides based N-terminal conservative part of surface Bac SGB of Ibc serotype and containing a first IgA-connecting site A with changed or native sequence MLKKIE, polypeptide exhibits immunologically relevant and protective properties, and they also high selectively connect IgA.
12 cl, 20 dwg, 4 tbl, 19 ex
SUBSTANCE: invention concerns the method for making an immunogenic reagent which causes immune response on infection Bacillus anthracis, including one to several polypeptides which together represent three domains of a full-size protective antigen (PA) from B anthracis or their versions, and at least, one of specified domains contains domain 1 or domain 4 of the PA, or its version. Said polypeptides of specified immunogenic reagent, and the full-size PA are produced as a result of expression in a recombinant cell E.coli. The invention also discloses an expression vector and nucleic acid with percent of residual guanidine and cytosine more than 35%, coding immunigenic polypeptide which is said protective antigen (PA).
EFFECT: high-yield immunogenic polypeptide.
13 cl, 5 dwg, 3 tbl, 6 ex
SUBSTANCE: invention is related to production of new hybrid polypeptide GST-CFP10 by microbiological synthesis with properties of species-specific protein-antigen CFP10 Mycobacterium tuberculosis, which may be used for early species-specific diagnostics of tuberculosis infection. Recombinant plasmid DNA pTB232 has been constructed, which codes hybrid polypeptide GST-CFP10 with properties of mycobacterial antigen CFP10, with average molecular weight (m.w.) 3.4 MDa and having size of 5257 p.n. Recombinant strain of bacteria E. coli BL21/pTB232 contains recombinant plasmid DNA pTB232, is producer of hybrid polypeptide GST-CFP 10 with properties of mycobacterial antigen CFP10 and is deposited in KM GNC VB "Vector" under number B-1027. Recombinant polypeptide GST-CFP10, produced with strain of bacteria E. coli BL21/pTB232, contains as protein-carrier N-end polypeptide fragment glutathione S-transferase S.j. (226 a.o. with m.w. of 26.3 kDa), joined via end site of thrombin hydrolysis (LVPR∧GS) with C-end polypeptide fragment of antigen CFP10 (100 a.o. with m.w. of 10.8 kDa) and has complete aminoacid sequence with length of 326 a.o. and m.w. of 37.1 kDa, given in text of description.
EFFECT: use of invention provides for the possibility to produce target highly pure hybrid polypeptide GST-CFP10 in preparative amounts with preservation of immunogenic properties of the latter.
3 cl, 4 dwg, 4 tbl, 6 ex
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: biotechnology, microbiology, medicine.
SUBSTANCE: invention represents group of Neisseria proteins eliciting antigen properties. Protein eliciting antigen properties comprises fragment of protein ORF 40 (amino acid sequence of ORF 40 is given in the invention claim) that involves 7 or more conservative amino acids arranging in succession. Proteins as components of proteins in the claimed group are used as a medicinal agent or for it manufacturing for treatment and prophylaxis of infection caused by Neisseria, and for manufacturing the diagnostic preparation. Invention relates also nucleic acid encoding the Neisseria protein. Nucleic acid is used as the protein. Applying the invention provides the maximal recognition and reactivity between strains of Neisseria. Invention can be used in manufacturing curative-prophylactic preparations with respect to Neisseria meningitides.
EFFECT: valuable biological and medicinal properties of antigen.
27 cl, 51 dwg, 10 ex