Way of reception functionally active recombinant protein of ulcer (lf) lethal factor, recombinant plasmide dna pethis-lf coding active protein lf; h strain escherichia coli bl-hislf producing active protein of malignant anthrax lethal factor
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
The invention relates to biotechnology, specifically genetic engineering, relates to a method of obtaining the active recombinant protein lethal factor of anthrax LF, recombinant plasmid DNA IS-LF that encodes an active protein lethal factor of anthrax LF, and the strain of the bacteria Escherichia coli, producing an active protein lethal factor of anthrax.
Lethal factor of anthrax (LF), along with the protective antigen (PA), is an essential component of the lethal toxin of anthrax and is a major determinant of death in anthrax infection. The main areas of application of the active protein LF are of biological research, Biomedicine and drug development, in particular the development of diagnostic kits for early detection and pharmaceutical preparations for the treatment of anthrax infection and vaccination against anthrax. It may be important to combat local outbreaks of diseases and for rapid neutralization of the effects of possible bioterroristic acts using bacteria or bacteria spores of anthrax.
Anthrax is an infectious disease caused by toxigenic strains of gram-positive bacteria Bacillus anthracis. The virulence of the bacteria is determined cap is Ulai, consisting of poly-D-glutamates acid, and exotoxin, which includes three components - lethal factor (LF), and protective antigen (PA) and the swelling factor (EF). Individually, these proteins do not have a toxic effect, but the combination of LF+PA (lethal toxin) and EF+PA (edema toxin) lead to various pathological consequences as in mammals and man, and in cell culture. Functionally LF anthrax is a Zn2+sensitive metalloprotease, specifically cleave proteins of the family kinases mitogenactivated protein kinases MARCH (in particular, MEK), resulting in lysis of the cells exposed to the toxin under the influence of macrophages. Conducted studies on the enzymatic activity of LF, its substrate specificity, its three-dimensional structure.
Lethal factor of anthrax is a protein with a molecular mass of 90 kDa, which is structurally composed of four domains with a predominantly helical structure. Domain 1 consists of 12 spiral sections, balancing chetyrehyacheechny layer. This domain binds to the protective antigen. Domain 2 is similar with the second domain VIP2 toxin of Bacillus cereus, however, does not possess its catalytic radicals. Inside a sequence of domain 2 is the sequence of domain 3. This domain is made up of repeats that has grown up is in duplicate sequence, originally belonging to the domain 2. Domain 4 is catalytically active protease. Beta-layer and six alpha-helices form a structure similar to that which exists in other metalloprotease, thermolysin. The substrate binding protein of the family of MARCH happens in the recess between domains 3 and 4. The active center of the enzyme is composed of amino acid radical Glu687for the water molecule and atom of zinc, associated with His radical686, His690, Tight728and Glu735(Pannifer AD, Wong TY, Schwarzenbacher R, Renatus M, Petosa C, Bienkowska J, Lacy DB, Collier RJ, Park S, Leppla SH, Hanna P, Liddington RC. (2001) Nature, 414, 229-233).
There is a method of isolation of recombinant lethal factor of anthrax (Gupta P, Batra S, Chopra AR, Singh Y, Bhatnagar R. (1998) Infect Immun, 66(2), 862-865), which use expression design-based plasmids pQE30, freexpression in the strain E. coli SG 13009. The lack of production of the protein using this design is that more than 40% of the protein is in a degraded state (probably due to the cleavage by E. coli proteases), which, of course, reduces the yield of protein when using this design. High initial degree of degradation of the protein leads to that required three stages of protein purification to obtain a homogeneous product. In the used expression design does not contain sequences for specific detects and gene-expression product.
Known closest to the claimed genetic engineering is a method of obtaining mutant protein LF using design-based rat under the control of the promoter of phage T5 in the expression E. coli strains (RF patent No. 2287581, publ. 20.11.2006). Thus obtained protein and its mutants used for the development of vaccines against anthrax. However, it is known that recombinant protein LF synthesized with the use of used construction has up to 40% degradation. In the patented expression design does not contain sequences for the specific detection of gene-expression product.
The invention solves the problem of creating producer and obtain active recombinant protein lethal factor of anthrax with high yield, a low degree of degradation, easily detected and simplified technology using two chromatographic purification stages instead of three.
The problem is solved at the expense of recombinant plasmid DNA pETHIS-LF for synthesis of hybrid recombinant active protein lethal factor of anthrax in Escherichia coli cells, having a molecular weight of 5.15 Hmm, consisting of BamHI-NdeI fragment of DNA plasmids 22b(+); synthetic sequence that encodes six histidines and the peptide c-myc, as well as BamHI/SalI DNA fragment containing the modified depending on annoy to these sites to sequence full-lethal factor of anthrax, containing as a genetic marker gene β-lactamase determining the stability of the transformed plasmid pETHIS-LF cells of Escherichia coli to antibiotics penicillin; unique recognition sites of restriction endonucleases that are located in the following distance to the left from the BamHI site: XhoII 657 mon, StuI 1572 mon, XhoI 1625 mon, BcoI 1625 mo.
Also due to Escherichia coli strain BL-LFHIS, producing a hybrid polypeptide containing the lethal factor of anthrax, merged with the sequence of the six histidines and peptide C-myc.
And also owing to the method of production of recombinant lethal factor of anthrax, including the transformation of Escherichia coli cells BL21(DE3) expression plasmid DNA pETGST-LFmin that encodes a recombinant protein lethal factor of anthrax, culturing the obtained strain, disruption of bacterial cells by the addition of Triton X-100 in buffer solution of pH 7.4 containing proteiny inhibitor, followed by purification of the target product metal-chelate chromatography and gel filtration.
The technical result of the invention is to obtain in pure form and high yield (95% purity, 100 mg liter of bacterial culture without fermentation) homogeneous recombinant protein lethal factor of anthrax with simplified technology for two-stage chromate is graphy instead of three.
To improve products and reduce the degree of degradation of the protein LF as the basic design used a plasmid with T7 promoter polymerase contain a sequence corresponding to the peptide C-myc for detection of the product, and the sequence of the six histidines for effective purification of the synthesized protein. To obtain basic vector design pETNHIS in plasmid pET22b(+) (Novagen), not containing a BglII site (sequence restriction site violate the processing of the plasmid by restriction enzyme BglII, followed by completion of all fragment maple DNA polymerase I and legirovaniem completed end) restriction sites NdeI and BamHI introduce the DNA fragment synthesized by polymerase chain reaction coding sequence of the six histidines and the peptide C-myc.
Received as described plasmid pETNHIS on the sites of the restriction endonucleases BamHI and XhoI clone full gene lethal factor of anthrax, split by made by amplification of a fragment comprising the primers terminal sites of the restriction endonucleases BamHI and SalI (this is possible because when splenii restrictase XhoI and SalI form is compatible sticky ends).
The obtained expression design pETHIS-LF transform into E.coli strain BL21(DE3) and Express in 2xYT medium. The yield of recombinant protein years is a high factor of anthrax high purity (97%) after two steps of chromatographic purification reaches 15% relative to the total protein of the cell.
The resulting product detects specific antibodies to the peptide C-myc.
The proposed technical solution, use producing strains of Escherichia coli BLHIS-LF containing plasmid DNA pETHIS-LF and are superproduction recombinant protein LF.
Construction of recombinant plasmid DNA pETHIS-LF provides a high level of expression of the cloned gene in them lethal factor of anthrax.
Gene lethal factor of anthrax obtained polymerase chain reaction with DNA bacteria anthrax at the State Research Center for Applied Microbiology (fsri GNORPM).
The ends of the gene containing the corresponding applied to the vector of the sites of the restriction endonucleases BamHI and SalI entered using the polymerase chain reaction using synthetic oligonucleotide primers LFBamHI, LFSalI (figure 1), and then the gene cloned in the vector plasmid pETNHIS.
Offer producing strains of Escherichia coli BL-HISLF characterized by the following features.
Morphological features. Cells are rod-shaped form, risperadone, gram-negative.
Cultural characteristics. Bacterial cells grow well on simple nutrient media. When grown on agar, form round, muddy yellow-grey colonies with a slightly rough edge, diameter up to 3 mm During growth on liquid media (LB, 2xYT) form intensivo the dregs.
Physical and biological characteristics. Cells grow at temperatures from 4 to 40°C With an optimum of 37°C and optimum pH values from 7 to 7.5. As the carbon source used amino acids, carbohydrates, glycerol. As the source of nitrogen can be used as organic compounds (amino acids, yeast extract, tripton and others), and inorganic mineral salts in the ammonium form.
Resistance to antibiotics. Cells are resistant to the antibiotics penicillin (100 μg/ml).
Producing strains obtained by transformation of competent E.coli cells corresponding recombinant plasmid DNA.
Strain BL-HISLF is superproduction. When induction of isopropylthio-β-galactoside is effective synthesis of the target protein LF, which accumulates in the cytoplasm of cells in a soluble state and is up to 50% of the total protein of the cell.
The invention is as follows. Construct recombinant plasmid DNA pETHIS-LF, containing the full sequence of the gene lethal factor of anthrax. This full gene lethal factor of anthrax get polymerase chain reaction using synthetic oligonucleotide primers (figure 1). The primers contain the sites of the restriction endonucleases BamHI (N-end of the gene, primer LFBamHI) and SalI (con the gene, primer LFXhoI), the resulting DNA digested with appropriate restriction endonucleases and then are ligated with the cleaved sites BamHI and XhoI vector plasmid DNA pETNHIS. Figure 2 presents the sequence of a gene full lethal factor of anthrax. Figure 3 shows the sequence of the expression constructs plasmids pETHIS-LF.
Ligase mixture transform competent cells of E. coli DH12S or other cells that do not contain DE3 of lysogeny and their own resistance to the antibiotics penicillin, and plated on 2xYT-arap containing 100 µg/ml ampicillin or other penicillin antibiotic. The resulting clones examined for the presence of plasmids containing the gene LF polymerase chain reaction with primers LFBamHI and LFSalI. Of the clones, PCR which leads to the formation of the desired fragment length analysis of the reaction products agarose gel electrophoresis, isolated plasmid DNA by the method of alkaline lysis. Embedding fragment confirmed by sequencing of plasmid DNA. The obtained recombinant plasmid DNA to transform competent cells of the strain E. coli BL21(DE3), the resulting clones examined for the expression level of the target protein and selected producing strains.
Producing strains of E.coli BL-LFHIS grown on rich medium (2×YT, Terrific Broth, and others) to achieve an optical density of culture 0.6-1.2 TH inducyruut 1 mm isopropylthio-β-D-galactoside. The expression of spend 6 hours at a temperature of 25°C.
Cells of the expression of culture is harvested by centrifugation at 5000 rpm and treated with lysozyme (0.5 mg/ml) at 4°C in buffer containing 30 mm Tris-HCL pH 7.4, 150 mm NaCl, supplemented protease inhibitor (Complete EDTA-free protease inhibitor cocktail, Roches) by adding Triton X-100 to 0.5%. To the lysate add MgCl2up to concentrations of 5 mm and destroy DNA Dnazol (10 µg/ml). The resulting lysate centrifuged for 20 minutes at 18000 g and applied to the column. The first phase of a chromatographic purification is carried out on metal-chelate sorbent. The second cleaning stage is performed on the gel filtration column in buffer containing 30 mm Tris-Hcl pH 7.4 and 70 mm NaCl. The fractions containing purified LF, determined by denaturing polyacrylamide gel electrophoresis and frozen for storage at -70°C.
Detection of the product is carried out by immunoblot with antibodies specific to the peptide C-myc.
The activity obtained recombinant lethal factor of anthrax measured using specific substrate for lethal factor of anthrax and contains Hamamatsu (Lethal Factor Protease Substrate 2, the firm Biotrend, Germany) according to the absorption increase in the solution at a wavelength of 405 nm on a spectrophotometer. Kinetic measurements were carried out for 30 minutes at 37°C.
Invented the e illustrate graphics:
figure 1 - primers for the amplification of the gene lethal factor of anthrax;
figure 2 - sequence of a gene full lethal factor of anthrax and its fragment without domain I;
figure 3 - sequence expression constructs plasmids pETHIS-LF.
The invention is illustrated by the examples.
Construction of recombinant plasmid DNA pETNHIS-LF
Chemical synthesis of oligonucleotides perform solid-phase fostamatinib method on a DNA synthesizer ASM-102U (BIOSSET, Novosibirsk) with increasing oligonucleotide chain in the direction from 3' to 5' end using secure phosphamidon (5 dimethoxytrityl-N-acyl-2'-deoxynucleoside-3'-O-(β-Tianeti-diisopropylamino)-phosphites), activated tetrazolo. Synthesis is carried out at the scale of 0.5-0.7 μm, using as a carrier of porous glass (pore size 500 A), to which 3'-succinate connection joining the first nucleoside link (load 20-30 µm/g). Use synthetic cycle standard phospholidine method.
For preparation of vector DNA, 3 μg of plasmid pETNHIS treated with 20 μl of BamHI buffer (company New England Biolabs) and 10 units of the restriction enzyme BamHI, and then 20 μl of buffer 2 (New England Biolabs) and 10 units of endonuclease XhoI (company New England Biolabs). The restriction is carried out for 1 hour at 37°C. the Vector fragment length 6,09 KBP after electrop is cut in the agarose gel is placed in a dialysis bag with maximal bandwidth then 12-14 kDa and elute under the action of the current in Tris-borate buffer for electrophoresis, and then treated with a mixture of phenol-chloroform (1:1) and precipitate DNA from solution by ethanol.
For the preparation of a fragment of the gene LF carry out PCR amplification using as a matrix of bacterial DNA anthrax 0.05 μg in a sample, but as primers synthetic oligonucleotides a and b (20 each RM). 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 1 unit of activity of Pfu-polymerase (firm Promega). PCR is carried out in the regime providing for 1 minute denaturation at 95°C, annealing 30 seconds at 57°C, the elongation is 40 seconds at 72°C, 25 cycles of PCR. After that the reaction mixture deproteinizing a mixture of phenol-chloroform (1:2) and precipitated with ethanol. The precipitate was dissolved in 20 μl of water and digested with restriction endonucleases BamHI and SalI (reaction with restriction enzyme SalI production Fermentas held in the buffer Of Fermentas). The target fragment are elution from agarose gel.
0.5 μg of the obtained synthetic gene fragment recombinant lethal factor of anthrax was added to a solution of 0.2 µg described above vector and fragment are ligated in 10 μl of buffer containing 20 mm Tris-HCl pH 7.6, 10 mm MgCl2, 0.2 mm ATP, 10 mm dithiothreitol and 10 units of activity of T4-DNA ligase for 4 hours at 16°C.
An aliquot of the reaction mixture used to transform electropo what situation (device WITH 600, mode 129 Ohms, 2.5 kV) electrocompetent cells of E. coli DH12S. Transformants plated on plates with hot-agar containing 50 μg/ml ampicillin. Screening of recombinants performed using PCR amplification under the same conditions, and that obtaining a DNA fragment for cloning, using as a matrix of cells of a single bacterial colony containing the plasmid. Positive clones are identified by the presence of fragment length 2340 mo in the analysis of PCR mixture by agarose gel electrophoresis. Selected clones secrete DNA plasmids pETNHIS-LF, using the method of alkaline lysis.
Getting a producer strain E. coli BL-LFHIS and determine its productivity
Producing strains of E.coli BL-LFHIS get transformation electrocompetent cells E. coli BL21(DE3) with plasmid pETHIS-LF, as described in example 1. Single clones BL21(DE3)carrying plasmid pETHIS-LF, Express in analytical amounts (5 ml). For this purpose, E. coli BL-LFHIS grown on rich medium (2xYT, Terrific Broth, and others) to achieve an optical density of culture 0.6-1.2 OE and induce 1 mm isopropylthio-β-D-galactoside. The expression of spend 6 hours at a temperature of 25°C. Analyze the level of expression of a protein lethal factor of anthrax by denaturing polyacrylamide gel electrophoresis and immunoblot with antibodies against peptide C-myc. For electrophoresis equal Alik is the notes of different cell clones centrifuged at 5000 rpm for 5 minutes, precipitated cells are dissolved in 100 ál lyse buffer with dye bromophenol blue, treated with 20 seconds ultrasound, heated for 3 minutes at 100°C and applied on the gel. After completion of electrophoresis the gel is stained, Kumasi R-250 according to standard methods and scanned using a densitometer Shimadzu CS-930. For setting the immunoblot membrane Hybond C+ and the gel for 10 minutes, soaked in buffer containing 38.6 mm glycine, 47.9 mm Tris-base, 0,0385% sodium dodecyl sulfate, 20% methanol. The transfer is carried out at current 1 mA per cm of gel. Blocking of the membrane after transfer is 3% solution of bovine serum albumin in PBS pH 7.4 containing 0.05% Tween. The blocked membrane was incubated with antibodies to C-myc (clone CRL-1725, ATSS) at room temperature for 2 hours in a solution of PBS containing 0.5% BSA, and after washing the primary antibody with a mixture of PBS-0.05% Tween 20, the membrane is incubated overnight with peroxidase conjugate antibodies to mouse antibodies. The washed blot are using peroxidase substrate of chloronaphthalen. Clones giving the highest yield of protein, are the superproducers, they are grown on rich medium (2xYT) during the night, add glycerol to 15% and frozen at -70°C for storage.
For preparative expression strains superproducer E.coli BL-LFHIS grown over night at 37°C with the addition of 2% glucose, escaut 10 ml overnight culture in 1000 ml of 2xYT medium, containing 0.1% glucose and 50 μg/ml of ampicillin during, and grown at 37°C to an optical density of 1 OE. The grown culture is cooled to 25°C. add IPTG to 1 mm and spend the expression of 6 hours at 25°C. Next, the cells collected by centrifugation at 5000 rpm for 10 minutes and stored in the form of frozen precipitation at a temperature of -70°C.
The protein purification lethal factor, obtained using design pETHIS-LF, and determination of its activity
Cells of the expression of culture is suspended in a buffer containing 30 mm Tris-HCL pH 7.4, 150 mm NaCl, supplemented protease inhibitor (Complete EDTA-free protease inhibitor cocktail, Roches) and treated with lysozyme (0.5 mg/ml) at 4°C for 30 minutes. Are lysed cells by adding Triton X-100 to 0.5%. To the lysate add MgCl2up to a concentration of 1 mm and destroy the genomic DNA of E.coli by Dnazol (10 µg/ml). The resulting lysate centrifuged for 20 minutes at 18000 g and applied to a column of metal-chelating sorbents with immobilized ion cobalt (Talon, Clontech) in a buffer containing 20 mm Na2HPO4pH 8.3 and 70 mm NaCl. The column was washed with 10 volumes of the same buffer and elute the protein with a solution containing 200 mm imidazole in the same buffer. The second cleaning stage is carried out on a gel filtration column Superdex 200 in buffer containing 30 mm Tris-Hcl pH 7.4 and 70 mm NaCl. The fractions containing purified LF set the Ute by denaturing polyacrylamide gel electrophoresis, as described in example 2, and frozen for storage at -70°C. the Purity of Preparata is 95% according to densitometry. The yield of recombinant protein lethal factor is 30 mg per liter culture after a full cleanup.
Proteolytic activity of the obtained preparation is assessed by the cleavage of a specific substrate for lethal factor of anthrax containing Hamamatsu (Lethal Factor Protease Substrate 2, the firm Biotrend, Germany), the absorption increase in the solution at a wavelength of 405 nm on a spectrophotometer (ASAP). Kinetic measurements were carried out for 30 minutes at 37°C. the Kinetic parameters of selenia substrate obtained lethal factor of anthrax are kcat/KM=4.15±0.7-1μm-1.
1. Recombinant plasmid DNA pETHIS-LF for synthesis of hybrid recombinant active protein lethal factor of anthrax in Escherichia coli cells, having a molecular weight of 5.15 Hmm, consisting of: BamHI-NdeI fragment of DNA plasmids 22b(+); synthetic sequence that encodes six histidines and the peptide with the ICC, as well as BamHI/SalI DNA fragment containing adapted to these sites to sequence full-lethal factor of anthrax, containing as a genetic marker gene β-lactamase determining the stability of t is informirovannii the plasmid pETHIS-LF cells of Escherichia coli to antibiotics penicillin; unique recognition sites of endonuclease restriction placed on the following distance to the left from the BamHI site: XhoII 657 mon, StuI 1572 mon, XhoI 1625 mon, BcoI 1625 mo.
2. The Escherichia coli strain BL-LFHIS, producing a hybrid polypeptide containing the lethal factor of anthrax, merged with the sequence of the six histidines and the peptide with the ICC.
3. A method of obtaining a recombinant lethal factor of anthrax, including the transformation of Escherichia coli cells BL21(DE3) expression plasmid DNA pETGST-LFmin that encodes a recombinant protein lethal factor of anthrax, culturing the obtained strain, disruption of bacterial cells by the addition of Triton X-100 in buffer solution pH 7.4, containing proteiny inhibitor, followed by purification of the target product metal-chelate chromatography and gel filtration.
SUBSTANCE: there is disclosed leprosy microbacteria storage method consisting in low temperature effect on the infected substance. The stored substance is cooled at temperature -18°C that allows using household freezers.
EFFECT: simplified method of storage of M leprae.
FIELD: oil and gas industry.
SUBSTANCE: invention relates to receiving of exo-polysaccharides used in the capacity of thickeners during the operation of oil fields, particularly to method of receiving exo-polysaccharides of alginate type. Method provides cultivation of Azotobacter vinelandii "ИБ-1" on culture medium by Fedorov, containing mixture of microelements and molasses in amount 60.0 g/l in fermenter at conditions of aeration; 0.5 of air volumes per 1 medium volume per minute and mixing 300 revolutions per minute at temperature 25°C during 48 hours. Release of exo-polysaccharides by means of sedimentation by isopropyl alcohol in amount 70% by volume.
EFFECT: increasing of exo-polysaccharides yield.
1 tbl, 7 ex
FIELD: medicine; biotechnologies.
SUBSTANCE: method involves cultivation of strain-producer Penicillium canescens chosen from the group including: multicopy strain Penicillium canescens Abf6 (VKM № F-3935D), in gene abfA arabinoxylane-arabinofuranhydrolase Penicillium canescens containing arabinoxylanase and α-L-arabinofuranosidase activity, strain Penicillium canescens AbfB6 (VKM № F-3937D), in gene abfB2 α-L-arabinofuranosidase Penicillium canescens, multicopy strain Penicillium canescens AgLA33 (VKM № F-3936D), in gene aglA α-galactosidase Penicillium canescens containing both α-galactosidase and galactomannanase activity, strain Penicillium canescens AgLC4 (VKM № F-3934D), in gene aglC α-galactosidase Aspergillus awamori, multicopy strain Penicillium canescens FAE9 (VKM № F-3938D), in gene faeA feruloyl-esterase Penicillium funiculosum, multicopy strain Penicillium canescens XG9 (VKM № F-3939D), in gene xegA xyloglucanase Penicillium canescens.
EFFECT: extended range of enzymatic agents.
21 dwg, 1 tbl, 6 ex
SUBSTANCE: present invention relates to biotechnology and genetic engineering. Proposed here is a set of lux-biosensors, consisting of Escherichia coli cells obtained using recombinant DNA technology, containing plasmids with bacterial luxCDABE genes controlled by stress inducible promoters PgpoE and PgrpE and non-inducible Plac promoter.
EFFECT: invention can be used in evaluating degree of contamination of the environment by hydrophobic detergents.
SUBSTANCE: present invention relates to a new strain, which produces bleomycine A2 antibiotic, effective in treatment of squamous cell carcinoma and a range of other types of tumours. The Streptomyces griseocarneus subsp.bleomycini All Russian collection of industrial microorganisms S887 strain is obtained from multiple-stage mutagenic effect of methylenenitroguanidine, UV-radiation and 60Co gamma rays on the initial strain N.1129 with subsequent extraction of mutants on media, containing aminoethylcysteine, carbamoylcysteine, canavanine and methylthreonine as selective agents. The obtained N. 7-99 strain is deposited in the All Russian collection of industrial microorganisms under number S887. Compared to the initial strain, this strain is more than ten times more active. The product produced by the strain is a single-component antibiotic. The content of the antibiotic complex in the culture liquid of the strain according to the invention is 140 mcg of mono-component bleomycine A2 in 1 ml. Purity of the target product is achieved through chromatography of eluate with "КБ-2" on a Styrosorb MN-200 sorbent, on which only impurities are sorbed.
EFFECT: high activity of the new All Russian collection of industrial microorganisms S887 strain defines its prospective use in industrial production of bleomycine A2.
2 cl, 1 tbl, 1 ex
SUBSTANCE: proposed strain of Nonomuraea roseoviolacea subsp carminata (Actinomadura carminata) BKIIM-S1081 is obtained as a result of multiple stage mutagenic effect of nitrimethylbiurets, methylnitrosoguanidine and gamma rays 60Co on the initial strain № All Russian collection of industrial microorganisms S-806 with subsequent extraction of mutants on media, containing streptomycin and daunorubicin as selective agents, and deposited under the number All Russian collection of industrial microorganisms S-1081. Carminomycine is obtained from biosynthesis of a strain producer, treatment of culture liquid with acetone and oxalic acid, alkalisation of the solution, extraction of antibiotic using chloroform, re-extraction of antibiotic using an aqueous buffer at pH=5.0, repeated extraction using organic solvents at pH=8.0, concentration of the extract, filtration through a neutral PTFE sorbent and extraction of antibiotic through precipitation from the solution in dichloromethane-hexane or petroleum-ether in form of a base.
EFFECT: increased output of carminomycine antibiotic, efficient in therapy of soft tissue sarcoma, lymphosarcoma, reticulosarcoma, chorionepithelioma, acute myeloblastoma and lymphoblastoma, chronic myeloid leukaemia (120 mcg in 1 ml of culture liquid).
2 cl, 1 tbl, 1 ex
SUBSTANCE: present invention relates to microbiology and biotechnology. Obtained is a recombinant strain of Yarrowia lipolytica which produces lipase, developed through transformation of the Yarrowia lipolytica Polf strain using integrative plasmid pZ-ura3d4-hp4d-LIP2.
EFFECT: obtained Yarrowia tipolytica strain can be used in obtaining lipase on an industrial scale.
2 dwg, 4 ex
SUBSTANCE: strain of mycelial fungus Aspergillus oryzae All Russian collection microorganisms F-3927 D - producer of acid α-amylase can be used in various areas of industry and agriculture for saccharifying starch-containing raw material.
EFFECT: increase of acid α-amylase output.
SUBSTANCE: invention relates to microbiology and plant science, and envisage new application of bacterium strain Bacillus subtilis 11B having broad antagonist activity towards phytopathogenic bacteria and fungi, aimed at increasing the germinating ability of seeds of gramenaceous plants in heavy-metal contaminated soils. The method is carried out by soaking seeds of gramenaceous plants in suspension of bacterium strain Bacillus subtilis 11B in concentration of 1.0×106-1×108 cells in 1 ml.
EFFECT: increase of the germinating ability of gramenaceous plants.
FIELD: microbiology; medicine.
SUBSTANCE: strain Amycolatopsis orientalis subsp.eremomycini All-Russian collection of industrial microorganisms Ac-807 is eremomycin producent and can be used as source of eremomycin in medicine for treatment of severe infections.
EFFECT: increase of eremomycin output.
2 tbl, 1 ex
FIELD: genetic engineering.
SUBSTANCE: invention refers to genetic engineering and can be used in medical and biologic industry for making recombinant heterocarpine that is an antagonist of human release factor of growth hormone (GHRH). There is disclosed complete nucleotide sequence coding polypeptide heterocarpine; there are disclosed the related primer sequences to be used in heterocarpine gene cloning, as well as genetic make-ups including specified sequence, particularly hybrid gene coding fused protein containing polypeptide heterocarpine, as well as expression vectors for said hybrid gene. There is described method for making recombinant heterocarpine as His-tag fused protein, providing application of the host cells transformed or transfected with the disclosed genetic make-ups.
EFFECT: recombinant heterocarpine according to the invention can be used in making a medicinal agent for cancer treatment.
9 cl, 6 ex
SUBSTANCE: peptide is obtained from phage peptide library including set of static peptides with length of 12 aminoacid residues, by affine selection of phage clones containing peptide capable of specific linking to antibodies of benzo[α]pyrene and benzo[α]anthracene. Peptide displays specific interaction effect on antibodies of benzo[α]pyrene and benzo[α]anthracene and features molecular weight of 1.3 kDa and registered aminoacid sequence LHLPHHDGVGWG encoded by nucleotide sequence SEQ ID NO:1.
EFFECT: application in medicine as a base for peptide medicine development for immunologic prevention of malignant tumours of humans.
4 dwg, 3 ex
SUBSTANCE: present invention relates to genetic engineering, more specifically to obtaining anticoagulative protein extracted from nematodes (NAP) and can be used in medicine. To obtain a medicinal preparation based on NAP, methanotrophic yeast host cells are cultured, encoding rNAPc2 or rNAPc2/praline, until attaining the desired cell density. NAP is then extracted from the said yeast host cells through cation-exchange chromatography on an expanding layer. To purify the NAP medicinal preparation, hydrophobic-interaction chromatography is used. NAP is extracted and purified at pH levels below 4.
EFFECT: simple and more efficient method of obtaining anticoagulation proteins from nematodes.
25 cl, 8 dwg, 7 tbl, 6 ex
SUBSTANCE: claimed are expression vectors for obtaining IL-21 in E.coli cells. IL-21 coding nucleotide sequence, included in composition of novel vectors, contains modifications aimed at optimisation of codons and secondary mRNA structure for translation in E.coli. As a result of transformation with claimed vector structures E.coli strains suitable for industrial scale application have been obtained. Methods of wide scale IL-21 production which use said strains have been elaborated, allowing to obtain more than 1g/l of recombinant cytokine.
EFFECT: novel compounds possess useful biological properties.
14 cl, 1 dwg, 12 tbl, 19 ex
SUBSTANCE: hybrid protein - human insulin precursor consists of N-end fragment of human gamma-interferon connected through peptide linker with amino acid sequence of human proinsulin. Recombinant human insulin is obtained by cultivation of Escherichia coli JM109/pHINS11 strain-producer, carrying plasmid pHINS11, isolation of inclusion bodies and their dissolving in buffer which contains urea and dithiotreitole. Then hybrid protein re-naturation, sedimentation of admixture compounds, purification of re-naturated hybrid protein by ion-exchanging chromatography, combined fermentative hydrolysis of hybrid protein with tripsin and carbopeptidase B are carried out. At the last stage insulin purification with cation-exchanging chromatography and method of highly efficient reverse phase liquid chromatography are carried out.
EFFECT: simplification of obtaining highly purified recombinant human insulin and increase of its output.
6 cl, 1 dwg, 4 tbl, 5 ex
FIELD: pharmacology; biotechnology.
SUBSTANCE: invention concerns biotechnology area, in particular to the gene-engineering way providing mass production multimeasured recombinant human Mannan-Binding Lectin (MBL), also can be used in the biomedical industry. The offered way includes a) a cultivation stage of cells-owners lines CHO, transfectant with a recombinant vector pMSG-MBL, containing sequence of human MBL coding area, and b) a clearing stage of the recombinant protein. Thus at the first stage preferably use new cellular line CHO MBL D1-3, deposited with the Korean collection of sample cultures at number KCTC 10472BP which is cultivated in a protein-free medium with bioreactor use, and on the second selective clearing of high-molecular form MBL of medium cultivation is spent, providing separation of samples with the help anion exchange chromatography, their drawing on a column with an immobilised MBL-binding protein on it , in particular with glycosilated protein of a cover of a virus pre-S, in the presence of ions of calcium and the subsequent elution using a buffered solution with EDTA or EGTA.
EFFECT: high output of functionally active product opens possibilities of its application by working out of therapeutic agents for treatment of virus, bacteriemic or fungoid infections.
6 cl, 11 dwg, 9 ex
SUBSTANCE: invention relates to biotechnology, in particular to production of hormones and can be used for culturing invertebrates. Gonadotrophin, selected from the invertebrate Asterina pectinifera is a peptide with molecular weight 4500-4900, it has two subunits, the protein structure of which is combined with SS-bridges, formed between residues of SH cysteine contained in the subunits.
EFFECT: interfusion and oxidation of these two subunits after synthesis allow producing gonadotrophin having gonadal promoting activity.
4 cl, 8 dwg, 1 tbl, 2 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, in particular to hepatic cells production, and may be used in medical science. From the whole liver or resected part thereof, a cell population enriched with living cells of human liver, including hepatic stem cells/precursor cells, is obtained. Cell population contains functional hepatocytes and biliary cells expressing cytokeratin 19 (CK19), but not expressing albumin, as well as hepatic stem cells/precursor cells 9 to 13 mcm in diameter and expressing EP-CAM, CD 133 markers. Resulting cell population is used for hepatotherapy.
EFFECT: production of living population of hepatic cells sufficiently efficient for regeneration.
60 cl, 16 dwg
FIELD: medicine; biotechnologies.
SUBSTANCE: adenoviral vector carrying in the genome structure a human lactoferrin gene, administer into an allantois of the 9-10 day chicken embryoses. The subsequent planting is performed by egg incubation at temperature of 37°C within 70-75 hours. Then allocate the recombinant protein from the allantoic liquid of a chicken embryos.
EFFECT: depression of expenses and obtaining simplification of the recombinant human lactoferrin.
FIELD: medicine, biotechnologies.
SUBSTANCE: invention can be used for obtaining of the factor VII of blood coagulation. Derivatives of a polypeptide of the factor VII with amino-acid replacements Q250C, R396C and P406C are obtained or with Cysteinum attached to the S-end of native sequence of the factor VII. Obtain derivatives with use of transgene technologies in eucariotic cells-owners of mammals.
EFFECT: invention allows obtaining derivatives of the factor VII with the kept activity of the coagulative factor VII and with increased ability conjugate with PEG, in comparison with the natural form of a polypeptide.
20 cl, 2 dwg, 8 ex
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