Recombinant phage m13 dna pol t7 and recombinant strain of phage m13 - producer rna polymerase of phage t7
(57) Abstract:Usage: biotechnology, genetic and protein engineering. The inventive by introducing a DNA vector phage MMR unique BamH1 restriction site Sau3A-Sau 3A fragment of plasmid PAR 1219, containing the gene for RNA polymerase of phage T7, constructed recombinant DNA M13polT7, on the basis of which the resulting strain of phage M13 PMBC PH 714 for synthesis of DNA polymerase T7 exit 2106units act/g wet biomass. 2 S. p. f-crystals., 2 Il. The invention relates to biotechnology, in particular genetic engineering, and is a recombinant phage DNA M13polT7, containing the gene for RNA polymerase of phage T7 and strain of phage M13polT7 producing RNA polymerase of phage T7.RNA polymerase of phage T7 is widely used to obtain RNA probes for blot-hybridization; obtaining biologically active RNA, studying the processing of eukaryotic RNA exon-intron mapping of genomic DNA  in Addition, the enzyme can be used for labeling RNA with radioactive or biotinylated of nucleosidase, and in certain systems for sequencing nucleic acids 
However, the strain of E. coliHMS174 transformed this loft is">Recently as cloning vectors have been actively using filamentous phages of E. coli. Created various vector derivatives of phage: M13, fd and fi  the Advantage of vectors of this type is that filamentous phage during infection is not are lysed bacterial cells, and chopinot replicative form ragovoy DNA in the cell reaches 200-300 molecules that delivers a high dose of the cloned gene, and hence a sufficiently high yield RNA polymerase of phage T7. In addition, phage DNA is easier to allocate and use compared to plasmid DNA.Known phage M13polT7-1, used in a vector system capable of expression of foreign genes in E. coli cells under the control of a late promoter of phage T7.This vector system is based on the separate introduction of genes into bacteria by pre-transformation of E. coli cells with plasmid with the expressed gene with subsequent infection in their bacteriophage M13polT7-1, which acts only as a carrier of the gene for RNA polymerase, but is not its producer.Known recombinant plasmid PAR1219, containing the gene for RNA polymerase of phage T7.The technical task proposed is the construction of recombinant phage M13polT7, providing an increased synthesis of the enzyme RNA polymerase of phage T7 in bacterial cells.Phage DNA M13polT7 containing the DNA fragment encoding the RNA polymerase of phage T7 has a molecular weight of 6.6 MDA (size 9949 N. p.) and consists of the following elements:
vector BamHI fragment of phage M13mp10, size 7244 N. p.Sau3A-Sau3A fragment of plasmid PAR1219, containing the gene for RNA polymerase of phage T7, size 2705 N. p.plac-lacuv5 promoter;
a unique restriction site: BamHI;
marker gene: lacZ.The strain of phage M13polT7 has the following cultural and morphological properties.Morphology of negative colonies on solid medium: forms clear plaques with a diameter of 2 mm.Functional properties: nelisiwe phage.The optimum growth: (37+1)oC on YT environment.The strain deposited at the all-Union collection of industrial microorganisms Institute "Vniigenetika" number VKPM PH 714.The essence of the invention lies in the fact that the DNA fragment encoding the RNA polymerase of phage T7, injected into the phage vector DNA of filamentous phage M13. The obtained recombinant phage provides a synthesis of the RNA polymerase of phage T7 in cells of E. cI injected fragment from plasmid PAR1219  encoding RNA polymerase of phage T7.The resulting phage M13polT7 in E. coli cells provides a synthesis of the RNA polymerase of phage T7 (25-30% of total protein).Thus, for the first time obtained phage DNA, which infected her cells E. coli fusion protein with the properties of RNA polymerase of phage T7, due to gene expression RNA polymerase of phage T7.In Fig.1 shows the schematic construction of phage M13polT7 with the following symbols: polT7 gene RNA polymerase of phage T7, SD binding site of the ribosome, plac-lacuv5 promoter, lacZ part galactosidase gene; Fig.2-densitogram electrophoretic separated lysates of E. coli cells infected with phage M13polT7. The shaded peak corresponding protein polT7.The essence of the invention disclosed in the following examples.Example 1. Method of constructing recombinant phage M13polT7. 1 μg DNA vector phage M13mp10  decompose the restriction enzyme BamHI (10 units) in a buffer containing 10mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM MgCl2,1mM dithiothreitol. The reaction is carried out for 1 h at 37oC. analysis of the completeness of the hydrolysis is carried out using electrophoresis. The drug deproteinized using phenol extraction, the DNA precipitated with ethanol and resuspended in TE-buffer containing 10mM Tris-HCl pH 8.0 and 1 mM EDTA is up with Sau3A hydrolysis of plasmid pAR1219  the Selection of individual fragment is carried out by electrophoretic separation in a 6% polyacrylamide gel followed by Electrosila.Ligation mixture hydrolyzed phage M13mp10 (0.1 µg) and fragment encoding gene RNA polymerase (0.1 ág),performed using DNA ligase of phage T4 (4 units) in a volume of 30 ál (incubation are within 10 h at 12oC).The resulting phages that do not have a blue color on indicator plates from Ygal (0.1 ág/ml) and IPTG (1 Mm) analyze restriction fragments length polymorphism analysis of replicative forms of these phages using the restriction enzyme HaeIII.Recombinant phage DNA, which has in its composition the fragment containing the gene for T7 RNA polymerase in the correct orientation under the control of the lacuv5 promoter), marked M13polT7.Example 2. Gene expression RNA polymerase of phage T7 in the composition of the phage M13. To study the gene expression RNA polymerase of phage T7 E. coli cells D5F infecting phages: M13polT7 and M13mp10. E. coli cells D5F pokasivaut to optical density (OD) of 0.6-0.7% (590 nm) in LB medium at 37oC. Then contribute phage M13polT7, and in the control experiment, the phage M13mp10; and an inductor - IPTG (isopropyl-D thiogalactoside) (0.1 mm). The ratio of the cell: phage - 1:1 (multiplicity of infection).Suspension cultured for 6 h, which allows to obtain the maximum yield of the target protein in the case of phage M13polT7.On electrophoregram separation browarnik vector phage M13mp10.Determination of enzyme activities carried out, using as specific template DNA of phage T7.Based on the data of electrophoresis level of product RNA polymerase synthesized by recombinant phage, approximately 25-30% of the total protein.Example 3. The selection and titration of the phage. To the culture fluid after separation of infected cells add 0.25 to the original volume of PEG solution (containing polyethylene glycol 6000 20 g, NaCl 10 g, water to 100 ml) and left for 15 min at 20oC. the Phage was separated by centrifugation (15 min) and resuspended in a buffer solution containing 20 mm Tris-HCl pH 7.5, 20 mm NaCl, 1mm, ADEA (1/10 of the original volume). Titration of phage performed by standard methods. Usually get the drugs phage with a title 10-10 PFU/ml.Example 4. Purification of recombinant RNA polymerase of phage T7. E. coli cells DH5F infected with the phage M13polT7, precipitated by centrifugation. 6 g of biomass suspended in 160 ml of 50 mm Tris-HCl buffer pH of 7.9,containing 2 mm EDTA, 0.1 mm of dithiothreitol, 200 mm NaCl, 5% glycerol and 10 mg of lysozyme. Cells destroy ultrasound for 1 min and centrifuged for 30 min at 1000 rpm To the supernatant add solution streptomycinresistant to a final concentration of 0.2% at 0 rpm for 20 min.The supernatant containing the RNA polymerase of phage T7, add ammonium sulfate to 50% saturation. The suspension is centrifuged for 30 min at 10000 rpm dissolved in 40 ml of 10 mm potassium phosphate buffer pH 8.0, containing 10 mM-mercaptoethanol, 1 mM EDTA, 12% glycerine (buffer A) and 0.15 M NACl, and subjected to dialysis against the same buffer for 12 hThe desalted enzyme solution is applied on a column (1.51.5) phosphocellulose P11.RNA polymerase of phage T7 elute column with a gradient of NaCl (0.25 M To 0.5 M) in the buffer And RNA polymerase eluted at NaCl concentration of 0.25 M To 0.4 M. the Fractions containing the enzyme are combined and applied to a column (1,5x6 cm) with blue dextran-separate. The column is washed once with 40 ml of buffer a containing 0.25 M NaCl, then with 20 ml of buffer a containing 1 mm GTP and 1 mm ATP.The enzyme elute 0.55 M potassium phosphate buffer pH 8.0, containing 1.5 mm dithiothreitol, 40 mm EDTA and 12% glycerol.The fractions containing RNA polymerase of phage T7, collect and cialiswhat against buffer A, and then concentrate against 10 mm potassium phosphate buffer pH 8.0, 10mM b-mercaptoethanol, 50% glycerol and 180 mm KCl.Thus, in the scheme of purification receive 12106units of the act. homogeneous RNA in the S="ptx2">With 1 g of wet biomass receive 2106units of the act. homogeneous enzyme.References:
1. KKrieg,P. A. And Melton,D. A. Methods Enzymoll.155,397(1987).2. Axelrood,V. D. Rramer,F. R. Biochemistry 24,5716(1985).3. Davanloo,P. et al.Proc.Natl.Acad.Sci:USA 81,2035(1984).4. Messing, J. and Vieira, J., Gene,1982,v.19.p7269-276.5. A. A. Il'ichev, N. In.Melamed, A. I. Zakabunin and other DAN, 1988,I. 303,N2,c. 496-498.6. Single-atranded DNA Phages (1978). eds.Dehardt D. T. Dressler D. H. and Ray, D. S. Cold Spring Harbor Laboratory,ID 11724 Cold Spring Harbor,New York. 1. Recombinant phage M13 DNA polT7 sized 9949 p. O. molecular weight of 6.6 Hmm, consisting of a Bam HI DNA fragment phage vector M13 mp 10 size 7244 p. O. Sau Sau 3A 3A DNA fragment size 2705 p. O. from plasmid pAR 1219 encoding RNA polymerase of phage T7; plac lacuv 5 promoter,
and containing marker gene lac Z; unique restriction site Bam HI.2. Recombinant strain of phage M13 PMBC PH 714 producer RNA polymerase of phage T7.
FIELD: biotechnology, in particular prephenate dehydrotase-chorismatmutase and DNA fragment encoding the same.
SUBSTANCE: prephenate dehydrotase-chorismatmutase is isolated from Methylophilus methylotropus and may contain replacements, deletions, inserts, or incorporations of one or more amino acids. Said enzyme plays an important role in L-phenylalanine biosynthesis. Method of present invention makes it possible to improve L-phenylalanine production due to increased activity of enzymes involving in L-phenylalanine biosynthesis pathway.
EFFECT: improved L-phenylalanine production.
2 cl, 2 dwg, 1 tbl
FIELD: biotechnology, in particular method for production of L-amino acid except L-glutamic acid.
SUBSTANCE: claimed method includes cultivation of bacteria Methylophilus, which is capable to grow utilizing methanol as a main carbon source and to produce L-amino acid; and collection L-amino acid from culture. For example, bacteria Methylophilus with increased activity of dihydrodipicolinate synthase and aspartokinase is used. Said activity is increased by introducing DNA encoding dihydrodipicolinate synthase which is not inhibited with L-lysine by negative back coupling, and DNA encoding aspartokinase which is not inhibited with L-lysine by negative back coupling, into cells.
EFFECT: increased yield of L-amino acids.
10 cl, 7 dwg, 6 tbl, 7 ex
FIELD: genetic engineering, molecular biology, biochemistry.
SUBSTANCE: recombinant plasmid DNA pTES-His-OPH is constructed for expression of polypeptide eliciting properties of organophosphate hydrolase comprising Cla I/Hind III fragment of plasmid pTrcTEGF, fragment of plasmid pTES-OPH with nucleotide sequence that encodes amino acid sequence of the matured form of organophosphate hydrolase, and nucleotide sequence encoding 6 histidine residues that is located by 5'-end of nucleotide sequence encoding organophosphate hydrolase. Based on indicated plasmid the strain Escherichia coli TSKMIBKH-29 - a producer of polypeptide eliciting properties of organophosphate hydrolase is obtained. Applying the invention provides preparing polypeptide with properties of organophosphate hydrolase by simplified technology and this polypeptide elicits the improved catalytic effectiveness of action with respect to thio-containing phosphoric acid triesters. Invention can be used for carrying out hydrolysis of organophosphate compounds.
EFFECT: valuable biochemical properties of producer.
2 cl, 4 dwg, 2 tbl, 4 ex
FIELD: medicine, genetics, biochemistry.
SUBSTANCE: invention relates to new NOS-variants or mutants that comprise structural modifications in site Akt-dependent phosphorylation. Modified NOS-proteins or peptides, in particular, human proteins or eNOS-peptides having change of amino acid residue corresponding to S/T in motif of the consensus-sequence RXRXXS/T of NOS-polypeptide of wild type and nucleic acid molecules encoding thereof can be used in genetic therapy and proteins and NOS-peptides can be used in screening methods of agents modulating activity of NOS. The advantage of invention involves the creature of new NOS-variants or mutants that can be used in genetic therapy.
EFFECT: valuable medicinal properties of mutants.
25 cl, 1 tbl, 9 dwg, 3 ex
SUBSTANCE: invention relates to mutant carbamoyl-phosphate synthase from Escherichia coli wherein amino acid sequence in 947-951-positions of natural carbamoyl-phosphate synthase in replaced with any amino acid sequences from SEQ ID NO:1- SEQ ID NO:9. Similar mutant carbamoyl-phosphate synthase from Escherichia coli containing any deletions, insertion, substitutions, or additions of one or more amino acids in one or more positions excluding 947-951-positions is described. Also disclosed are DNA fragment encoding aforementioned mutant carbamoyl-phosphate synthases, as well as method for production of carbamoyl-phosphate derivatives using strain Escherichia coli modified with said DNA fragment. Further invention relates to various strains Escherichia coli, for instance: strain Escherichia coli 311 that is producer of orotic acid; strain Escherichia coli 333 that is producer of L-arginine and citrulline; strain Escherichia coli 374 that is producer of citrulline.
EFFECT: method for production of carbamoyl-phosphate derivatives such as L-arginine, citrulline, pyrimidine derivatives in increased amounts as compared with natural strains Escherichia coli.
10 cl, 2 dwg, 5 tbl, 4 ex
FIELD: biotechnology, biochemistry, microbiology.
SUBSTANCE: method involves to a method for preparing the fodder complex enzyme preparation possessing activities of endo-1,4-β-glucanase, β-glucanase, endo-1,4-β-xylanase, phytase, pectin-lyase and α-galactosidase. The preparation is prepared by combined culturing multicopy strains of fungus Penicillium canescens VKM F-3868D and VKM F-3869D. The enzyme preparation possessing activities of endo-1,4-β-glucanase, β-glucanase, endo-1,4-β-xylanase, phytase and pectin-lyase is prepared in combined culturing multicopy strains of fungus Penicillium canescens VKM F-3868D and VKM F-3870D. For preparing the enzyme preparation possessing activities of endo-1,4-β-glucanase and β-glucanase the strain Penicillium canescens VKM F-3868D is cultured, and for preparing the enzyme preparation possessing activities of phytase, pectin-lyase and α-galactosidase the strain Penicillium canescens VKM F-3869D is cultured. The enzyme preparation possessing activities of phytase and pectin-lyase is prepared by culturing the strain Penicillium canescens VKM F-3870. The claimed invention provides expanding assortment of enzyme preparations.
EFFECT: improved preparing method.
12 cl, 3 dwg, 12 ex
FIELD: biotechnology, gene engineering, ecology.
SUBSTANCE: Escherichia coli cells containing plasmides with luxCDABE-genes are produced by recombinant DNA method under control of PkatG, PsoxS, PrecA, PgrpE inducible stress promoters. On the base of the same kit of Escherichia coli bacterial cells is developed for heptyl detection in environment medium.
EFFECT: accelerated method for heptyl detection.
1 tbl, 4 dwg
SUBSTANCE: invention relates to method for DNA amplification, method for cloning of second DNA and method for DNA reverse transcription. In each method heat stable DNA polymerase is used which encoded by SEQ ID NO:7, derived from Anaerocellum thermophilum or recombinant R.coli strain, transformed by vector containing SEQ ID NO:7. DNA polymerase catalyzes matrix-targeted DNA polymerization, has 5'-3'-activity and reverse transcriptase activity, and has no 3'-5'-endonuclease activity in presence of magnesium ions and in absence of manganese ions. DNA polymerase retains at least 90 % of its activity after incubation for 30 min at 80°C in absence of stabilizing detergents and has apparent molecular mass between of approximately 96 kDa and approximately 100 kDa. Magnesium-dependent reverse transcriptase activity of DNA polymerase is more than 30 % of DNA polymerase activity, and manganese-dependent reverse transcriptase activity of said polypeptide is more than 60 % of DNA polymerase activity.
EFFECT: increased precision of matrix RNA transcrption at high temperatures.
7 ck, 5 dwg, 7 ex
FIELD: medicine, peptides, biochemistry, pharmacy.
SUBSTANCE: invention relates to modification of glycosylation of proteins for preparing polypeptides with improved therapeutic indices including antibodies with enhanced antibody-dependent cellular cytotoxicity. For preparing indicated polypeptides cell-host is used that is modified with nucleic acid encoding enzyme β-1,4-N-acetylglucosaminyltransferase III (GnTIII). Prepared polypeptide represents, in particular, IgG or its fragment. Invention discloses a method for preparing polypeptide and antibodies or its fragment and a fusion protein prepared by indicated method. Invention describes a pharmaceutical composition used for increasing Fc-mediated cellular cytotoxicity and comprising antibody and carrier, and its using in cancer treatment, and a method for treatment of disease associated with elevated amount or production of B cells using indicated antibody, in particular, against CD20, and representing antibody IDEC-C2B8 in the preferable variant. Invention provides preparing polypeptide and antibody possessing the enhanced Fc-mediated cellular cytotoxicity that decrease the content of B cells in a patient body.
EFFECT: improved preparing method, valuable medicinal properties of polypeptide and antibodies.
38 cl, 21 dwg, 4 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention refers to genetic engineering and biotechnology and can be used in food industry. Enzyme chosen from gamma glutamyl hydrolase, GTP cyclohydrolase, dihydroneopterin aldolase and 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase is superexpressed in lactobacillus used in enzyme method for production of monoglutamylpholate and in composition of food, including dairy product.
EFFECT: higher pholate content in bioavailable form.
9 cl, 5 dwg, 2 ex
SUBSTANCE: composition is used for producing vaccines for preventing infectious diseases to occur, treating allergy and malignant neoplasm cases. Various invention embodiments comprise virus, virus-like particle, viral capsid particle, fag or their recombinant forms covered with any desired antigen highly ordered and having repetitions that is achieved owing to specific interactions taking place. Multi-functional process based on cassette-type system (alpha-vaccine techniques) allows one to produce antigen-coated viral particles like hepatitis B virus or measles virus.
EFFECT: causing marked immune response.
44 cl, 7 dwg
FIELD: medicine, genetic engineering.
SUBSTANCE: invention proposes a method that involves construction of bacteriophage library of random peptides based on oligonuleotide fragments encoding their, selection of bacteriophages binding with target-cells but not binding with cells of other types that can be involves in pathological process or able to show effect on its diagnosis and therapy, and confirmation of specificity of selected bacteriophages by using combination of different tests. Oligonucleotide fragments encoding random peptides are prepared by reaction of reverse transcription by using random primers and total RNAs isolated from indicated target-cells and cells of other types. Applying this invention provides preparing bacteriophages binding with target-cells with high degree of selectivity. Invention can be used in diagnosis, therapy and pharmaceutical industry.
EFFECT: improved preparing method.
3 cl, 2 dwg, 8 ex
FIELD: genetic engineering, virology, pharmacy.
SUBSTANCE: invention proposes the recombinant modified virus OF VACCINE Ankara able to express structural antigens of hepatitis C virus. Virus comprises DNA sequences encoding structural antigens of hepatitis C virus or their functional regions or epitopes of hepatitis C virus structural antigens. Also, invention proposes a pharmaceutical composition comprising such virus, eucaryotic cell infected with such virus, a method for preparing such virus and a method for preparing hepatitis C virus structural polypeptides. Invention can be used in virology and medicine for preparing hepatitis C virus antigen.
EFFECT: valuable properties of virus.
20 cl, 14 dwg, 1 tbl
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: genetic engineering, virology, medicine.
SUBSTANCE: invention relates to method for production of modified Vaccinia virus Ankara (MVA). Claimed method includes contamination of mammalian continuous cell line with Vaccinia virus Ankara (MVA) of wild type, followed by viruses cultivation and collection. Further fresh cells of the same cell line are infected with newly formed viruses. Abovementioned steps optionally are repeated. Also disclosed are strains of modified Vaccinia virus Ankara (MVA) and utilization thereof. Said strains are capable to growth in continuous cell lines.
EFFECT: strains having decreased virulence in relates to mammalians.
20 cl, 5 tbl
FIELD: genetic engineering, virology.
SUBSTANCE: invention proposes a method for preparing the attenuated recombinant strain of the influenza virus A.Method involves preparing the recombinant NS segment of influenza virus A comprising the functional RNA-binding domain and modification of its gene sequence below nucleotide 400 in the gene NS1. This modification prevents translation of the remaining moiety of the gene NS1. Also, invention proposes the genetic-engineering strain of influenza virus A, vaccine comprising thereof, and the recombinant segment of the gene NS of influenza virus A. The prepared genetic engineering strain of influenza virus A possesses the interferon-inducing effect and retains the interferon-nonsensitive natural phenotype and able to induce response as production of interferon in MDCK cells and in fertilized chicken eggs, and to grow in latter. Invention can be used in medicine and virology.
EFFECT: improved and valuable properties of recombinant virus.
17 cl, 20 dwg, 6 ex
FIELD: gene engineering.
SUBSTANCE: recombinant fragmid DNA pHEN-TAB, containing unique human single-strand antibody gene is selected from constructed in vitro combinatorial phage library udder controlling of lactose operon promoter. Then Escherichia coli HB2151 cells are transformed with obtained fragmid DNA to produce recombinant bacterium strain Escherichia coli HB2151/pHEN-TAB as producer of human single-strand antibody capable of binding of human tumor necrosis factor alpha. Said antibody gas affinity constant of Kaf = 3.96±0.52x108 M-1.
EFFECT: new soluble human single-strand antibody scTAB against human tumor necrosis factor alpha with high affinity.
3 cl, 6 dwg, 6 ex