Method for predicting tuberculous infection, recombinant plasmid dna coding the synthesis of cfp10-esat6 hybrid protein out of mycobacterium tuberculosis, the method of its obtaining, cfp10-esat6 hybrid protein and its application

FIELD: biotechnology, medicine.

SUBSTANCE: the present innovation deals with CFP10-ESAT6 hybrid protein that is able to induce Mycobacterium tuberculosis-specific hypersensitivity reaction of delayed type. This hybrid protein contains complete protein CFP10 out of M.tuberculosis combined with complete protein ESAT6 out of M.tuberculosis through linker amino acid sequence. It is, also, [presented a recombinant plasmid DNA pTBD16 for expression of hybrid protein CFP10-ESAT6. The latter should be obtained due to applying DLT1270 E.coli strain transformed with the obtained recombinant plasmid DNA pTBD16. The innovation suggests to apply the above-mentioned protein as a diagnostic kit of tuberculous infection both in mammalians and human beings. The method for predicting tuberculous infection has been revealed. Tuberculous infection should be predicted due to induction of hypersensitivity reaction of delayed type in case of subcutaneous injection of hybrid protein. Application of the present innovation enables to obtain a Mycabacterium tuberculosis-referring diagnostic kit, at specificity being 100%, sensitivity - 80%, not less.

EFFECT: higher accuracy and efficiency of diagnostics.

6 cl, 4 dwg, 4 ex

 

The invention relates to the field of medicine and biotechnology, specifically to the specific methods of diagnosis of tuberculosis and the new diagnosticum, allowing this way: a hybrid protein that is specific only to .tuberculosis obtained by using constructed in vitro recombinant plasmid DNA.

Research conducted by the world health organization (who)showed that the problem of tuberculosis (TB) still is extremely sharp, as evidenced by the level of infection in the population of the globe, accounting for more than 1 billion people. About 8 million people become ill with TB every year. While 44% are sick pulmonary form of the disease. About 2 million people die each year, which amounts to 23% of deaths worldwide and more than 50% mortality in African countries (Dye C, Scheele S, Dolin P, Pathania V and Ravinglione M, 1999, JAMA, 282, 7, 677-686).

To control the spread of TB and significantly reduce the risk of spread of the disease is necessary, including the creation of effective reagents for specific diagnosis.

Basic laboratory methods for the detection of tuberculosis infection for many years is the Mantoux test.

The basis of this method is the determination of delayed-type hypersensitivity against antigenic the determinants of mycobacteria. Reagent for the production of skin samples represents the total extract antigens from Mycobacterium bovis, which implies a very low level of specificity of the PPD test. A positive test does not detect differences between active form of the disease, prior sensitization by contact with M. tuberculosis, BCG (BCG) vaccination or cross-sensitization to other species of mycobacteria. Moreover, the antigenic components of the sample are not standardized and therefore samples from different sources may differ in skin reactions (Mustafa A S, T-cell subsets and cytokines interplay in infectious diseases, pp.201-211). Thus, there was the urgent need to identify antigens of M.tuberculosis, who may become candidates for the production of improved vaccines with universal efficacy, and specific diagnostic reagents for the detection of TB.

The development of biotechnology, in particular the use of technology DNA cloning and expression, has greatly facilitated the identification of several major antigens of M.tuberculosis.

Based on some of the identified antigens of M.tuberculosis (mostly proteins "heat shock", such as hsp18, hsp60, hsp70 and secreted proteins-antigens) have developed methods for the diagnosis of delayed-type hypersensitivity (GST) (see, for example, patent US 6458366, patent-similar - published avca Russia No. 98105682, Compounds for diagnosis of tuberculosis and methods of their use. Patent WO 0021983 TV vaccine and diagnostic based on antigens from M. tuberculosis cell. Patent US 6120776 Diagnostic skin test for tuberculosis).

However, a common drawback of these approaches is the lack of complete specificity of the used protein, polypeptide including hybrid reagents against M.tuberculosis, since such proteins are polypeptides are other mycobacteria strains and the vaccine strain BCG. In the case of the latter, the developed approaches are not suitable for the differential diagnosis of infectious immunity from vaccination.

In recent years, as a result of the comparative analysis of the genomes of different organisms, it was found that M. tuberculosis provides a synthesis of the two secretory protein genes esat6 and CFP10 that are absent in M. bovis and most non-pathogenic bacteria (A.S. Mustafa, 2001, Carrent Pharmaceutical Biotechnology, 2, 157-173).

On the basis of peptides protein genes esat6 was created specific diagnostic system that allows you to confidently differentiate the delayed-type hypersensitivity (GST) post-vaccination origin and GST that appear during infection with M.tuberculosis (Patent WO 00262248 Tuberculosis diagnostic test)is accepted by the applicant as a prototype.

Based on the proposed diagnosticum method was developed for the diagnosis of tuberculosis infection by induction of the reaction repercussive the major delayed-type incubation of T-lymphocytes predominantly peripheral blood of a patient in vivo or in vitro with a mixture of the above antigens. In case of positive reakciei using sensitive immunological methods were recorded elevated synthesis γ-interferon.

This method is very complicated in the production, requires expensive equipment and reagents, and therefore not suitable for mass application as screenimage method.

As stated in the description, peptides genes esat6 protein can be obtained including genetic engineering methods, however, describe a particular genetically engineered structures in the application materials no.

The present invention is to develop a specific relatively inexpensive and simple method of testing tuberculosis on the basis of domestic diagnosticum.

The task is solved in that in vitro constructed recombinant plasmid DNA (RTR)encoding the hybrid protein synthesis CFP10-genes esat6 of M.tuberculosis, consisting of a DNA fragment of the vector plasmid pQE30 containing the replicon ColE, gene β-lactamase, which determines resistance to ampicillin, lac promoter, and a DNA fragment encoding the fusion proteins CFP-10 genes esat6 and, beginning with the initiating ATG codon, a sequence of 6-his-tag residues, flanked by sites for restricted BamHI and kpni restriction sites containing between genes and CFP10 genes esat6 a nucleotide sequence encoding the site of hydrolysis by enterokinase.

Patented one the same way to obtain the hybrid protein CFP10-genes esat6 by cultivation of the strain DLT1270 E. coli having lac1 gene in the chromosome of the transformed recombinant plasmid DNA (D16))that encodes a hybrid protein CFP10-genes esat6, destruction of cultured cells in a buffer solution by ultrasound, subsequent selection of the hybrid protein from purified Taurus on and leaching Taurus inclusion method legendable chromatography.

Patent-pending method for the diagnosis of TB infection based on the proposed diagnosticum is the induction of hypersensitivity reactions of the delayed type and is polinom introduction to the patient an aqueous solution of the hybrid protein CFP10-genes esat6 of .tuberculosis in effective dose, and then (after 12-48 hours) a positive evaluation of tuberculous process is carried out visually by the diameter of erythema in 10-15 mm.

Construction of recombinant plasmid DNA, a method of obtaining a hybrid protein is as follows.

To construct the recombinant plasmid DNA was used expression vector pQE30, allowing to Express the proteins with the addition of 6 HIS N-end.

Schematic diagram of the structures:

1. pQE-cfp

ATG-6xHIS-(BamHI)-EK-CFP10-(Cloned)

2. pQE-cfp-esat

ATG-6xHIS-(BamHI)-EK-CFP10-(Cloned)-EK-genes esat6-(HindIII)

EK - site actions enterokinase, DDDDK, cuts after K.

Gene cfp10 was amplified DNA .tuberculosis lines H37Rv using nucleotide primers CFP-F and CFP-R having the structure sites of hydrolysis for the endonucleases BamHI and Kpn1 (see 1)

The obtained fragment was treated with BamHI+kpni restriction sites and cloned between the BamHI and kpni restriction sites sites of pQE30.

Recombinants were selected using restricting analysis. The selected clone Q30-cfp provided a fusion protein of the expected size (about 15 KD on PAGE).

Gene esat-6 was amplified DNA M.tuberculosis lines H37Rv using nucleotide primers ESAT-F and ESAT-R, having in its structure the sites of hydrolysis for the endonucleases BamHI and HindIII (see figure 2).

The obtained fragment was treated with kpni restriction sites+HindIII and cloned between the kpni restriction sites and HindIII sites Q30-cfp.

Recombinants were selected using restricting analysis. The selected clone Q30-cfp-esat was provided by the synthesis of a hybrid protein CFP-ESAT expected size (about 27 KD on PAGE).

Ligase mixture containing selected recombinants transformed strain DLT1270 E. coli-derived DH10B with l1 gene in the chromosome.

Night culture were seeded at 1:100 in LB-agar and grown on a shaker at 37°until OD 600=0.4 are. 3 Quiroga contributed inductor expresii (IPTG) to 0.05 mm and continued incubation for 3 hours.

From wet biomass, buffer containing 0.02 M Tris-HCl, 0.3 m NaCl, 2 mm PMSF, pH 8.0 by centrifugation after UV destruction of cells get washed calf inclusions which contain most purified fraction of the hybrid protein; a method of multistage legendable chromatography receive the main faction of the hybrid protein with vyhoda is 65% and with a purity not less than 98% (determined by electrophoresis on a Lammy in a 13% SDS page, see Fig 4).

Additional purified hybrid protein fraction obtained from leaching Taurus enable repetition of the operations provided for protein purification from Taurus enabled.

The invention is illustrated using graphic materials:

- figure 1 contains a pair of primers for cfp10-gene;

- figure 2 contains a pair of primers for esat 6 gene;

- figure 3 shows the molecular weight of the hybrid protein obtained by the method of electrophoresis;

- 4 - data on purity allocated hybrid protein according to electrophoregram by Lammy 13% SDS page.

The invention is illustrated by the following examples.

Example 1.

Construction of recombinant plasmid DNA (pTBD16) in vitro.

For preparation of vector DNA plasmid pQE30 (3 μg, 1 pmol) treated with 40 μl of buffer R (10 mm Tris-HCl, pH 8.5, 10 mm MgCl2, 100 mm KCl, 0.1 mg/ml BSA) restriction enzyme BamHI, and then 40 μl of buffer (50 mm Tris-HCl, pH 7, 5, 10 mm MgCl2, 100 mm NaCl, 1 mm DTT, 0.1 mg/ml BSA) with the restriction enzyme kpni restriction sites (10 edict.) for 1 h at 37°C. Vector fragment size of 3.7 KBP after electrophoresis in 1% agarose gel electrophoretic move in the layer of DEAE-paper, then elute 1M NaCl and precipitate DNA from solution by ethanol.

For the preparation of a fragment of the gene CFP-10 are amplification by PCR, using as template the chromosomal DNA of M. tuberculosis (0,01 µg / sample), and in which the quality of the primers - synthetic oligonucleotides CFP-F and CFP-R (60 pmol each). PCR is carried out in a DNA-amplifier, a buffer solution containing each of the four dNTP at a concentration of 0.5 mm and 5 edict. Taq-DNA-polymerase, as follows: denaturation 1 min at 94°C, annealing 30 sec at 60°With elongation of 40 s at 72°C, 30 cycles of PCR. After that the reaction mixture deproteinizing chloroform, evaporated to dryness, the residue was dissolved in 20 μl of water, then break down the same restrictases that were used in the preparation of the vector and produce the target fragment from the agarose gel.

The obtained synthetic fragment with the gene CFP-10 in the amount of 2 pmol was added to a solution of 1 μg of the above described vector fragment in 10 μl of buffer (20 mm Tris-HCl, pH 7,56, 10 mm MgCl2, 0.2 mm rATP, 10mm dithiotreitol) and are ligated with 10 edict. T4-DNA-ligase for 12 hours at 10°C.

An aliquot of the reaction mixture used to transform competent cells of E.coli DLT1270. Transformants plated on plates with YT-agar containing 50 μg/ml ampicillin. The obtained recombinant plasmid pQEcfp were treated with restrictase BamHI and HindIII cloning of genes esat6 gene. Gene genes esat6 was amplified from the DNA of M. tuberculosis H37Rv using primers ESAT-F and ESAT-R. the Obtained synthetic DNA fragment in the amount of 2 pmol was added to 1 μg of the above, etc) the RA pQEcfp and are ligated with 10 edict. T-4 DNA ligase for 12 hours at 10°C. an Aliquot of the reaction mixture used to transform competent cells DLT1270. The result was obtained plasmid D16.

Example 2.

Getting a hybrid protein genes esat6-CFP10.

Induction of protein synthesis.

Received nightlife culture DLT1270 E.coli (derived DH10B with lac1 gene in the chromosome)carrying plasmid D16.

Night culture were seeded at 1:100 in LB-agar and grown on a shaker at 37°C to OD600=0.4 are. Then made IPTG to 0.5 mm and continued incubation for 3 hours. The synthesis of the target protein was monitored using SDS page electrophoresis, comparing the spectrum of proteins before and after induction. Watched the fusion protein of the expected size: 27 kD (corresponds to the data Berthet, et al., 1998 on mobility and cfp10 genes esat6 in PAGE). Cm. Figure 3.

Example 3.

Technology selection CFP10 protein-genes esat6.

Protocol isolation of recombinant CFP10 protein-genes esat6 from biomass producer strain E. coli method legendable chromatography.

Buffer solutions.

A. 0.1M Tris-HCl buffer, 6M guanidinium, pH 8.0.

Century M Tris-HCl, 0.3M NaCl, 2 mm PMSF, pH 8.0

C. M Tris-HCl buffer (6M urea, M NaCl and M imidazole, pH 8.0.

D. M Tris-HCl buffer (6M urea, M NaCl and 0.3M imidazole, pH 8.0.

E. M Tris-HCl buffer, 6M urea and M NaCl, pH 8.0.

F. 0.1M Tris-HCl buffer, 0.5M NaCl and 2 mm EDTA, pH 8.0.

G. M Tris-HCl buffer, M NaCl, pH 8.0.

Training with the bent to the work.

1 ml of commercial iminodiacetate-sepharose Chelating Sepharose CL-6B (Amersham Pharmacia Biotech) is placed in Column Linbro Plastics column (ICN, catalogue number 158690) and washed from preservative (20%ethanol) 10 ml deionized water (hereinafter all processes in the column are held by gravity). Then passed through the column 2 ml (10 mg/ml) with NISO4·7H2O (GOST 446574), after which the sorbent is washed with 10 ml of deionized water and balance a column of 5 ml of buffer A.

The column used for separation of protein from 150 ml of biomass.

Obtaining Taurus enabled.

1. To wet biomass of 150 ml of culture medium add 5 ml buffer, homogenized with ultrasound for 1 min (22-23 kHz, 100 W), the homogenate was poured into 1.5 ml plastic tubes type "Eppendorf and centrifuged at 10,000 rpm for 10 min in a tabletop centrifuge "Eppendorf S" or similar. The supernatant is collected separately.

2. The precipitate is suspended in 3 ml of buffer without PMSF and centrifuged again at 14000 rpm (30 min, +4° (C) in the centrifuge "Eppendorf MiniSpin" or similar. The procedure of washing the precipitate repeat 5 times. Washed calf inclusion carefully separated from the supernatant and stored at - 30°C.

The secretion of the protein from inclusion bodies.

1. Sediment Taurus inclusion of 150 ml of culture medium was dissolved in 5 ml of buffer While stirring on a magnetic stirrer for 2 h at room te is the temperature. The solution is centrifuged at 14000 rpm (20 min +20° (C) in the centrifuge "Eppendorf MiniSpin", the precipitate discarded, the supernatant is used to produce recombinant CFP10 protein-genes esat6.

2. The supernatant of claim 1. applied on the column with 1 ml of the prepared sorbent using an automatic pipette "Inpipe".

3. The column was washed with 10 ml buffer A, then with 10 ml buffer, causing the solution to the column using an automatic pipette "Inpipe" portions of 2 ml, not allowing the sorbent to dry.

4. Protein elute with 5 ml of buffer D in penicillin vial 10 ml

5. The eluate was diluted with 5 ml of buffer E, then cialiswhat (18 h, +40° (C) first against 1 l of buffer F, then twice against 1 l of buffer G (2×18 h, +4°).

6. Dialysate centrifuged at 4000×g (30 min, +4° (C), the precipitate discarded, the supernatant add sodium azide to 0.02% and stored at +4°C.

7. The supernatant measure the protein concentration by the method of ICA (calibration bovine serum albumin), the purity of the protein was determined by electrophoresis on a Lammy in a 13%SDS page.

Selection of recombinant CFP10 protein-genes esat6 from leaching Taurus enabled.

All washing Taurus enable diluted 2-fold with buffer A, centrifuged at 14000 rpm (20 min +20° (C) in the centrifuge "Eppendorf MiniSpin", the precipitate discarded, the supernatant is used to produce recombinant CFP10 protein-genes esat6, starting with step 2. Gave the e by the method.

Data on purity of the obtained product are listed on electrophoregram protein 13%SDS page by Lammy (see figure 4).

Example 4.

Testing diagnostic reagent on the basis of the hybrid protein genes esat6-CFP10 to determine the delayed-type hypersensitivity to M. tuberculosis.

As the model used Guinea pigs, which was intradermally senzibilizirani live M. bovis BCG, M.avium and M.tuberculosis H37RA in the amount of 107microbial cells in 0.2 ml of phosphate buffer pH 7,2.

At the sixth week after sensitization in a shaved area of the skin of experimental animals were injected intradermally at 2 µg of antigen in 0.1 PBS and 10 units of tuberculin, obtained from strains of M. bovis BCG, M.avium and M.tuberculosis. After 24 hours was estimated diameter of erythema formed in the injection.

td align="center"> a 9.5±1,5
Table 1.

Evaluation of delayed-type hypersensitivity in Guinea pigs using the skin test reagent containing recombinant CFP10 proteins-genes esat6 of M.tuberculosis.
AntigenThe diameter of skin reactions in Guinea pigs immunized
M. bovisM.aviumM.tuberculosis
PPD-t8,9±1,38,0±1,412,9±1,2
PPD-b12,1±1,39,8±1,5
PPD-a8,8±1,512,6±1,49,2±1,3
Hsp706,5±1,37,2±1,410,1±1,4
CFP10-genes esat60,5±1,00,6±1,011,2±1,3

As follows from the above results, a diagnostic reagent for skin tests based on the hybrid protein genes esat6-CFP10 allows you to reliably differentiate the delayed-type hypersensitivity caused by infection with M.tuberculosis, from reactions associated with vaccination immunity (BCG) and infection M.avium.

Next, a known diagnosis of tuberculosis infection by subcutaneous injection of a combination of antigens genes esat6 and CFP10-specific against M.tuberculosis and .Bovis. This shows the sensitivity (73%) and specificity (93%) indicated diagnosticum for humans compared to PPD (Laurens A.H. van Pinxteren, Clinical and Diagnostic Laboratory Immunology, March, 2000, page 155-160) (D1).

In parallel experiments on the model of Guinea pigs examined two main parameters characterizing skin test:

1. specificity and

2. the sensitivity.

The specificity of the test was evaluated in Guinea pigs infected .Bovis (BCG), M.avium, as these two types of mycobacteria induce immunological cross-reactions. M. bovis using it for the production of BCG vaccine, a M.avium abundantly present in the environment and very often infects humans, causing no clinical symptoms, but induces an immunological reaction, causing a positive PPD test. As a positive control was used Guinea pigs infected with M. tuberculosis. In each experiment were 20 animals. After setting the skin test positive reaction was assessed after 5 days as inflammation at the injection site larger than 5 mm, As shown by experiments, skin test (KP), a hybrid protein containing genes esat6-CFP10, gave a positive result only in animals infected with M. tuberculosis, whereas the Mantoux test was positive in all groups of animals. Thus, the specificity of KP was 100%, whereas the Mantoux test was positive in 60% in the case .avium and 80% in cases .Bovis (BCG).

The sensitivity of the test was evaluated on the model of Guinea pigs previously infected with different doses of M. tuberculosis. As we mentioned earlier, the use of individual proteins in the formulation of skin samples may degrade the sensitivity of the test due to a significant decline in the overall antigenic mass, available in CP. In the experiment used two groups of animals infected with two doses of Mycobacterium 40 animals in each group (10000 and 1000000 microbial bodies). Test sensitivity is titelliste KP carried out with drugs, contains

1. CFP10,

2. Genes esat6,

3. Genes esat6-CFP10,

4. tuberculin test.

The results of testing on animals with low-dose infection

No. samplethe number of animalspositively.sensitivity %
110440
210660
310880
41010100

The results of testing on animals with high dose infection

No. samplethe number of animalspositively.sensitivity, %
110660
210770
310990
41010100

Thus, the proposed diagnosticum according to its performance is better than that specified in D1 (specificity 100%, sensitivity of at least 80%), as well as by its technological, and hence the SRT is mestnym indicators it is preferred.

Get diagnosticum is a new substance, the biological activity of which (at least in quantitative terms) does not follow from the prior art.

1. Hybrid CFP10 protein-genes esat6, is able to induce specific against M.tuberculosis reaction of hypersensitivity of the delayed type, containing full CFP10 protein from M. tuberculosis, merged with a complete protein genes esat6 of M.tuberculosis through a linker amino acid sequence.

2. The use of a hybrid protein CFP10-genes esat6, containing full CFP10 protein from M. tuberculosis, merged with a complete protein genes esat6 of M.tuberculosis through a linker amino acid sequence, as diagnosticum tuberculosis infection in mammals and man.

3. The use of a hybrid protein CFP10-genes esat6 according to claim 2, characterized in that the quality of the linker sequence of the hybrid protein CFP10-genes esat6 contains the site of hydrolysis by enterokinase.

4. Method for the diagnosis of tuberculosis infection by inducing hypersensitivity reactions of the delayed type percutaneously CFP10 antigens and genes esat6-specific against M.tuberculosis, with subsequent positive evaluation of the tuberculosis process visually in diameter erythema 10-15 mm, characterized in that the CFP10 antigens and genes esat6 the patient is administered in an effective dose in the form of an aqueous solution of the hybrid proteins is CFP10-genes esat6, which are specific against M.tuberculosis, and contains full CFP10 protein from M. tuberculosis, merged with a complete protein genes esat6 of M.tuberculosis through a linker amino acid sequence.

5. Recombinant plasmid DNA pTBD16 for expression of the hybrid protein CFP10-M.tuberculosis genes esat6, representing the vector plasmid pQE30 containing the replicon ColE, lac promoter, the gene of resistance to ampicillin, the initiating ATG codon, a sequence of 18 nucleotides encoding the 6-his-tag residues, which quickly built a stretch of DNA that encodes CFP10 proteins and genes esat6, flanked by sites for enzyme BamHI and kpni restriction sites and containing between genes and CFP10 genes esat6 a nucleotide sequence encoding the site of hydrolysis by enterokinase.

6. A method of obtaining a hybrid protein CFP10-genes esat6 for the diagnosis of TB infection, including the cultivation of a strain of E.coli DLT1270 transformed with recombinant plasmid DNA pTBD16 for expression of the hybrid protein CFP10-genes esat6, the destruction of cells of the strain E. coli DLT1270 in buffer solution by ultrasound and secretion of the protein from purified Taurus on and from leaching Taurus inclusion method legendable chromatography.



 

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19 cl, 2 dwg, 4 tbl, 1 ex

FIELD: immunology.

SUBSTANCE: invention relates to immunoenzyme analysis and can be used for assay of von Willebrand factor. Method involves immunoenzyme analysis wherein monoclonal antibody 5C3 is used as an immobilizing antibody, and a mixture of biotin-labeled monoclonal antibodies 2H2 and 7D12 is used as a detecting antibody. Also, invention relates to monoclonal antibodies produced by the strain of hybridoma cultured cells Mus musculus L. and directed against von Willebrand factor, and to strains of hybrid cultured cells Mus musculus L. producing indicated monoclonal antibodies. Invention provides the development of highly sensitive method for assay of von Willebrand factor.

EFFECT: improved method for analysis.

9 cl, 1 tbl, 2 dwg, 3 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to a method for preparing bacterium pyocyanic anatoxin used in specific therapy and prophylaxis of infections caused by Pseudomonas aeruginosa (pyocyanic bacterium). Method involves culturing microorganisms in liquid nutrient medium comprising the following components per 1 liter of distilled water: citric acid, 4.9-5.0; sodium chloride, 0.9-1.0; magnesium sulfate, 4.9-5.0; ferrous sulfate, 0.04-0.05; asparagines, 0.9-1.0; glucose, 0.9-1.0; glycerol, 2.9-3.0 ml, balance, to the final concentration of pyocyanic bacterium, 14-15 billion of microbial bodies in 1 ml. Then the prepared biomass-containing cultural fluid is subjected for sterilization by autoclaving at 0.6-0.7 atm for 45-50 min followed by detoxification with formalin and anatoxin-containing cultural fluid is separated from biomass by filtration and sorbed its on aluminum hydroxide gel. Using the invention provides enhancing antigenic activity of toxic product and to standardize condition for culturing the producer.

EFFECT: improved preparing method.

1 tbl, 1 ex

FIELD: biotechnology, medicinal virology, medicine.

SUBSTANCE: invention describes isolated strain of human hepatitis B virus "S"-133 Ooh comprising genome of subtype adr that is deposited in the European collection of cellular cultures at № P 97121504 or P 97121505 or P 97121506 and encoding polypeptide of basic surface antigen of human hepatitis B virus. Abovementioned antigen comprises mutation in amino acid sequence (polypeptide), namely, methionine at 133 position is replaced with threonine. Invention discloses variants of isolated nucleic acids encoding the mutated polypeptide and variants of isolated nucleic acids encoding peptides showing properties of the indicated polypeptide. The description text gives nucleotide sequences of nucleic acid variants encoding abovementioned polypeptide and peptides. Also, invention describes variants of vectors used in cloning DNA fragments of mutant human hepatitis B virus comprising isolated nucleic acid and isolated nucleic acid bound functionally with RNA transcription promoter respectively and wherein each isolated nucleic acid encodes indicated polypeptide. Invention discloses methods for producing polypeptide and peptide and methods for preparing the purified polypeptide and peptide by using indicated vectors. Also, invention describes purified polypeptides and peptides of mutated antigen retaining antigenic properties of polypeptides of the natural human hepatitis B virus, and methods for preparing antibodies raised to them and anti-antibodies, among them, monoclonal antibodies. Also, invention discloses different methods for using abovementioned isolated nucleic acids, polypeptides and peptides for aim for diagnosis and treatment of diseases associated with human hepatitis B virus. Using the proposed invention provides the development of diagnosis schedules and treatment of diseases associated with the mutated human hepatitis B virus.

EFFECT: valuable medicinal properties of mutant virus.

66 cl, 2 dwg, 4 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to the strain Bacillus subtilis 0017 isolated from a sample of typical chernozem (black soil) in Bashkorstan Republic and maintained in the collection of microorganisms of Biology Institute of Ufa scientific center RAS. The registration number of the strain in collection is 0017. The strain exceeds the known natural strains with respect to accumulation level of surfactin. Invention can be used for preparing biogenic surface-active substance surfactin possessing the multiple biological activity.

EFFECT: valuable properties of bacterial strain.

1 tbl, 4 dwg, 6 ex

FIELD: biotechnology, medicine, in particular viral disease treatment.

SUBSTANCE: invention relates to recessive dividing retroviral vector useful in inhibition of wild-type retrovirus replication. Vector contains retroviral long terminal repeat sequences; retroviral packing signal; nucleotide sequence encoding (expressing) genetic antiviral agent; and optionally the second nucleotide sequence. Disclosed are method for production of said vector and reproduction thereof. Further isolated and purified nucleic acid (NA) molecule providing of selective advantage in regard to viral generation packing into virions is disclosed. Uses of retroviral vector in particular for specific antibody production are described.

EFFECT: new genetic antiviral agents generating prolonged and stable immunological responses in regard, for example, to AIDS and cancer viruses.

97 cl, 11 ex

FIELD: biotechnology, gene engineering, microbiology.

SUBSTANCE: invention relates to TUL4spCBD recombinant protein comprising TUL4 Francisella tulergenis protein, Gly-Ser spacer and cellulose-binding domain of CelD endoglucanase gen from Anaerocellum thermophilus. Said protein has TUL protein antigen properties and is capable of spontaneous binding to cellulose-containing sorbents. Described is recombinant plasmid pTUL4spCBD DNA of 4388 n.p. length, encoding TUL4spCBD recombinant protein. Abovementioned plasmid contains: artificial bacterial operon of TUL4spCBD recombinant protein, including promoter region of N5 bacteriophage earlier promoter (7-87 n.p.); TUL4spCBD recombinant protein gene (115-1113 n.p.); transcription terminator (1134-1230 n.p.); beta-lactamase bacterial operon (4183-3323 n.p. of complementary chain)), providing ampicillin resistance; ColE1-type bacterial site of replication initiation, providing plasmid replication in E.coli strain (4388 n.p.). Also discoised is M15[pREP4, pTUL4spCBD] Escherichia coli strain as producer of TUL4spCBD recombinant protein and method for production of purified, concentrated and immobilized TUL4spCBD recombinant protein from said strain by treatment of cell hydrolyzate supernatant from M15[pREP4, pTUL4spCBD] Escherichia coli with cellulose-containing sorbent. Method for production of specific antibodies against TUL4spCBD protein also is described. Said method includes animal immunization with TUL4spCBD recombinant protein immobilized onto cellulose.

EFFECT: method for high yield production of TUL4spCBD recombinant protein; simplified method for purification, concentration and immobilization of said protein.

5 cl, 1 dwg, 4 ex

FIELD: biotechnology, immunology, molecular biology, medicine, pharmacy.

SUBSTANCE: invention describes the isolated human antibody or its antigen-binding fragment able to bind the human tumor necrosis factor (TNF-α). Amino acid sequence is given in the description. Invention discloses nucleic acid encoding heavy and light chain of isolated human antibody. Nucleotide sequences are given in the description. Invention describes recombinant vector expressing variable region of heavy and light chains of isolated human antibody, Chinese hamster ovary cells CHO dhfr- carrying vector. Invention discloses a method for synthesis of isolated human antibody. The isolated human antibody or its antigen-binding fragment can be used as an active component of pharmaceutical composition used in treatment of disturbances when activity of TNF-α is harmful. Using the invention allows neutralization of effect of TNF-α in case when its activity is harmful. Invention can be used in medicine.

EFFECT: valuable medicinal properties of antibody, improved method for synthesis.

17 cl, 11 dwg, 17 tbl, 4 ex

FIELD: biotechnology, biochemistry, amino acids.

SUBSTANCE: L-glutamic acid is produced in culturing corynebacterium possessing ability to produce L-glutamic acid. This microorganism shows reduced ability for synthesis of trehalose or its absence as result of destruction of gene encoding trehalose-6-phosphate synthase, or gene encoding maltooligolysyltrehalose synthase, or by addition of mutations in these genes. Invention provides preparing L-glutamic acid with high degree of effectiveness.

EFFECT: improved method and enhanced effectiveness for preparing amino acid.

6 cl, 1 tbl, 3 ex

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