Recombinant plasmid dna rs va for expression of the ribozyme in eukaryotic cells

 

(57) Abstract:

The use of biotechnology, in particular genetic engineering. The inventive constructed recombinant DNA pCVA designed for efficient expression of a stable and biologically active ribozyme in eukaryotic cells and consists of vector plasmids pUC 1813 and MaeI-CfrI - fragment size 180 N. p. from the genome of adenovirus birds CELO, containing the gene virusassociated RNA. 1 Il.

The invention relates to biotechnology, in particular genetic engineering, is a recombinant plasmid pCVA designed for the transcription of the genes of the ribozymes in the composition of sequences of virus-associated PHK (VA PHK) adenovirus birds FAV1 (CELO) in eukaryotic cells.

Ribozymes are molecules PHK with catalytic activity. Currently, they are being increasingly used as antiviral agents and gene therapy.

For effective suppression of gene expression using the ribozymes is required for their molar excess over the substrate. This can be achieved by using a high level of gene transcription of the ribozyme in the cell and high stability of the ribozyme.

rasoi III [3] Ventura et. al. [9] have introduced the gene of the ribozyme in the composition of the gene VAI PHK of human adenovirus. However, in the proposed design of the ribozyme lost enzymatic activity due to the negative influence of the secondary structure of the molecule VAI PHK of human adenovirus. The authors had to introduce additional sequences, providing posttranscriptional shortening of the 5'-end of the molecule, which led to the recovery of the enzymatic activity of the ribozyme. And this is largely complicated genetic structure.

The technical effect of the invention is the creation of plasmids designed for efficient expression of a stable and biologically active ribozyme in the culture of eukaryotic cells.

Molecular weight plasmids pCVA equal 952,4 KD (size 2860 N. p.)

This plasmid contains a unique site between the promoter boxes gene VA PHK CELO, which can be used to clone genes of ribozymes for their further transcription in the culture of eukaryotic cells.

The technical effect is achieved using plasmids pCVA, which allows it to be used as a carrier of the ribozyme molecule VA PHK adenovirus birds CELO.

The invention is illustrated che is.

Mae I Cfr I fragment of the genome of adenovirus birds CELO size 180 N. p. containing the gene virusassociated PHK [6]

Example 1. Construction of plasmids pCVA.

Method of constructing a plasmid containing the gene, is that DNA plasmids pUC1813 [5] is treated with a restriction restriktsii Sma I and mixed with individual Mae I Cfr I fragment of the genome of adenovirus birds CELO, pre-treated fragment maple to turn the protruding DNA ends in a blunt. The mixture is treated with DNA ligase of phage T4 and used to transform cells of E. coli DH5 Transformants plated on medium with ampicillin (resistance to ampicillin due to the genetic marker gene (b-lactamase), plasmid DNA from bacterial colonies analyzed by specific endonucleases and select the clone that contains the vector plasmid pUC1813 [5] with the insertion of a fragment of the genome of adenovirus birds CELO length 180 N. p. Definition pervichnoi the structure of DNA recombinant plasmids pCVA sequencing method Singer [8] confirmed the presence of recombinant plasmid pCVA gene sequence VA PHK CELO, which coincides with the previously published sequence of this gene [6]

Example 2. Cloning IDA pCVA.

Cells of E. coli DH5 a, containing plasmid DNA pCVA, grown in 200 ml of broth to title 10ocells/ml Cells precipitated by centrifugation (5000 g, 5 min, +4oC) and resuspended in 35 ml of a solution containing 8% sucrose, 0.5% of Triton X-100, 50 mm EDTA pH 8.0. Next, add 2.5 ml of freshly prepared lysozyme, quickly stirred and heated in a boiling water bath for 3 minutes, the Lysate centrifuged (20 000 g, 30 min, +4oC) DNA from the supernatant precipitated by an equal volume of isopropanol, the precipitate is collected by centrifugation (12 000 g, 20 min, +4oC) and resuspended in 5 ml buffer (10 mm Tris-HCl pH 8.0). Final purification of plasmid DNA carried out by the method of equilibrium centrifugation in CsCl density gradient with bromide by ethidium.

1 μg DNA plasmid pCVA incubated with restriction enzyme Kpn 2 I in buffer containing 33 mm Tris-acetate, 10 mm magnesium acetate, 66 mm potassium acetate, and 0.5 mm dithiothreitol. The reaction is carried out for 1 h at 55oC. deproteinized by the method of phenol extraction, the DNA precipitated with ethanol and resuspended in the buffer for T4 DNA ligase containing 66 mm Tris-HCl, pH 7,6 mm MgCl2, 5 mm dithiothreitol, 1 mm ATP.

DNA artificial gene ribozyme against mRNA reporter gene SEAP is rment per 1 ág DNA. The incubation is conducted for 10 h at 12oC.

The mixture transform cells of E. coli DH5 a, as follows: 0.1 ml of cell suspension of E. coli DH5 a make in 20 ml of nutrient broth LB and grown to a titer 3108cells/ml Cells are harvested by centrifugation (5000 g, 10 min, 0oC), suspended in 10 ml of 50 mm CaCl2and incubated for 30 min at 0oC. the cells are Then re-centrifuged (5000 g, 10 min, 0oC), resuspended in 1 ml of 50 mm CaCl2. 100 μl of this suspension are used for transformation. Blend the United DNA fragments incubated with the treated cells for 1 h at 0oC and 2 min at 42oC. After 20-fold dilution with LB broth transformed cells pokasivaut 1 h and plated on agar medium with ampicillin (100 µg/ml).

Plasmid DNA isolated from ampicillinulbactam colonies grown after 24 h, analyzed by hydrolysis with restriction enzyme Kpn 2 I. Selected plasmid DNA, designated as pCVArib3, containing in its composition oligodeoxyribonucleotide ribozyme gene size of 50 N. p. Final analysis of DNA recombinant plasmids and the determination of the orientation of the ribozyme gene relative to the promoter boxes VA PHK CELO carried out by the method of sequ oticheskih cells.

DNA plasmids pCVArib3 injected into the eukaryote cell line 293 [4] For this purpose, cells grown on minimal medium Needle with the addition of 5% fetal calf serum. Cells scatter for 24 h before transfection. A buffer containing 5 g/l HEPES, 8 g/l NaCl, and 0.37 g/l KCl, 0.125 g/l Na2HPO42H2O, 1 g/l glucose, pH 7,1, mixed with DNA plasmids and added dropwise 2.5 M CaCl2(50 μl/ml). The mixture is incubated at room temperature for 30 min and the resulting precipitate contribute to the culture medium at a rate of 1 ml of the precipitate in 10 ml of medium. 6 μg of the Plasmid carrying the gene of the ribozyme ([CVArib3), used for transfection 3105cells in 3 ml of culture medium.

48 h after transfection of the cells secrete the total fraction of PHK by previously described methods [7] 6 µg PHK annealed with excess [-32P] dATP-labeled specific primer, which is complementary to the sequence of the ribozyme. Then hold revertto reaction [7] using AMV reverse transcriptase -42oC, 30 minutes Radioactively-labeled products of this reaction are separated on 10% polyacrylamide gel with 8 M urea gel radioautographic. Experience has shown that there was a lengthening of the specific primers at 50 N. what exactly RT confirms transcription in eukaryotic cells gene, a ribozyme, which is integrated into the VA PHK CELO. The transcription level can be estimated as 10 pkg of ribozyme on 1 µg of total PHK.

Example 4. Inhibition of transient gene expression of SEAP using the ribozyme is transcribed as part VA PHK CELO, in cell line 293.

DNA plasmids pCVArib3 and pBc12/RSV/SEAP [2] is introduced into the eukaryote cell line 293 [4] as described in example 3, 1 µg DNA plasmids that produce SEAP (pBc12/RSV/SEAP), and 5 μg of the plasmid carrying the gene of the ribozyme (pCVArib3) used for transfection 3x105cells in 3 ml of culture medium. Determination of the enzymatic activity of SEAP carried out after 48 h after transfection according to previously described methodology [2]

The result of the experiment revealed a 50% reduction in enzymatic activity of SEAP in the culture fluid of cells nontransferring DNA plasmids pCVArib3.

Thus, the invention allows to obtain a high level of biologically active ribozyme in the culture of eukaryotic cells. Plasmid pCVA can be used for further cloning and transcription in eukaryotic cells genes ribozymes directed to the inhibition of viral genes and cellular oncogenes.

Literature

1. Zakharchuk A. N. Doronin, K. K"ptx2">

2. Berger, J. Hauber, R, Geiger, R. Cullen, B. R, Gehe, 1988, 66, 1 - 10.

3. Cotten, M, Birnstiel, M. EMBO J. 1989, 8, 3861 3866.

4. Graham, F. L., Smiley, J. Russel, W. C. Nairn, R. J. Gen. Karpova et al., 1977, 36, 59 72.

5. Kay, R. MePherson, J. Nucleic Acids Res. 1987, 15, 2778.

6. Larsson, S. Bellet, A. Akusjarvi, G. J. Virol. 1986, 58, 600 - 609.

7. Sambrook, J. Fritsch, E. E. Maniatis, T. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

8. Sanger, F. et al. Proc. Natl. Acad. Sci. USA 1977, 74, 5463.

9. Ventura, M. Wang, P. Ragot, T. Perricaudet, M. Saragosti, S. Nucleic Acids Res. 1993, 21, 3249 3255.

Recombinant plasmid DNA RS VA for expression of the ribozyme in eukaryotic cells, having a molecular weight of 952, 4 KD (size 2860 N. p.), consisting of a DNA vector plasmid pUC 1813 size 2680 N. p. and May I Cfr I fragment of the genome of adenovirus birds CELO size 180 N. p. including gene virus-associated (VA) RNA containing a unique restriction sites: two sites Hind III, Pst I, Sal I, Xba I, Bam HI and one site Sph I in the vector pUC 1813 and unique cloning site PKK 21 in the gene VA RNA CELO, genetic markers plasmids: bla gene.

 

Same patents:

The invention relates to biotechnology, in particular genetic engineering, is a recombinant strain of vaccinia virus, causing the synthesis of structural proteins of the virus Venezuelan encephalomyelitis of horses (VAL) in infected cells and protective immunity against VAL have them vaccinated laboratory animals, as well as the method of construction of this strain

FIELD: biotechnology, molecular biology, biochemistry.

SUBSTANCE: invention relates to regulatory sequences. Method involves isolation of DNA molecule with nucleotide sequence SEQIDNO:2 or SEQIDNO:3 that is necessary for expression of the required encoding sequence. Then vector comprising any of indicated sequences and the required sequence is constructed followed by transformation a plant with the prepared vector. Invention provides preparing transgenic plants with regulating expression of the required gene.

EFFECT: improved preparing method.

19 cl, 1 tbl, 6 ex

FIELD: biochemistry.

SUBSTANCE: the present innovation deals with an anti-sense oligonucleotide or one of its derivatives which can inhibit expression of human eg5 protein being relative to kinesin of motor proteins. The oligonucleotide has got a sequence being correspondent to that of nucleic acid coding certain part of human eg5. This innovation deals with the way to obtain the above-mentioned oligonucleotides, pharmaceutical composition for inhibiting human eg5 and its application. Advantage of the innovation deals with developing e new preparation to be applied for inhibiting cell proliferation.

EFFECT: higher efficiency of inhibition.

11 cl, 1 dwg, 2 ex, 3 tbl

FIELD: gene engineering, in particular purification and isolation of polynucleotides.

SUBSTANCE: invention relates to purification and isolation of polynucleotides regulating mammalian gene transcription and is useful in regulation of heterologous polynucleotide expression, obtaining transgene animals, and identification of affined regulatory DNA sequences. DNA containing transcriptional regulatory DNA of hamster gene EF-1α was isolated by screening of genome library to Chinese hamster ovary (CHO-K1). Chimeric polynucleotide including isolated regulatory DNA of hamster gene EF-1α operably bonded to gene sequence encoding target protein product other than protein encoded by hamster gene EF-1α was constructed. Obtained chimeric polynucleotide is used as component of expression plasmid for transformation or transfection of host cell. To increase target gene transcription in host cell DNA containing regulatory DNA of hamster gene EF-1α was integrated into host cell genome DNA in site operably bonded to target gene. Method of present invention make it possible to increase mRNA expression level for operably bonded heterologous polynucleotides by 3-11 times.

EFFECT: increased mRNA expression of operably bonded heterologous polynucleotides.

31 cl, 3 tbl, 7 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to polynucleotide encoding zwal gene product containing polynucleotide sequence selected from group including a) polynucleotide encoding polupeptide with amino acid sequence with at least 90 % identity to amino acid sequence represented in SEQ ID NO:2; b) polynucleotide which is complementary to polynucleotides from a), as well as primer representing polynucleotide containing at least 15 sequential base pairs of abovementioned polynucleotide.

EFFECT: new zwal gene encoding ionic zwal product.

6 cl, 1 dwg, 1 tbl, 5 ex

FIELD: biotechnology, biochemistry, amino acids.

SUBSTANCE: invention describes a polynucleotide showing activity of glucose-6-phosphate isomerase and comprising polynucleotide sequence taken among the group including: a) polynucleotide encoding polypeptide that comprises amino acid sequence identical at least by 90% with amino acid sequence represented in SEQ ID NO:2; b) polynucleotide that is complementary with polynucleotides given in sub-paragraph a). Also, invention describes a method for enhancing the metabolism intensity in pentose phosphate cycle by attenuation of pgi gene and a method for preparing L-amino acids. Invention provides preparing L-amino acids with the high effectiveness degree.

EFFECT: improved preparing method, valuable properties of polynucleotide.

16 cl, 7 dwg, 3 tbl, 6 ex

FIELD: molecular biology, criminology, genetic medicinal trials.

SUBSTANCE: the present innovation deals with new markers , the method for their obtaining and applying to identify one's sex in DNA-containing human biological samples. The innovation suggested enables to detect chromosomal abnormalities by sex more accurately and at high sensitivity.

EFFECT: higher accuracy and efficiency of identification.

4 cl, 3 dwg, 3 ex, 2 tbl

FIELD: biotechnology, biochemistry, genetic engineering.

SUBSTANCE: invention proposes a method for construction of genetically modified strains of microorganisms able to destroy steroids. These strains comprise multiple inactivated genes, for example, genes encoding enzymes steroid dehydrogenases implicated in destroying the steroid ring. The gene kstD1 is an example of such genes. Strains comprising the multiple amount of inactivated genes encoding enzymes destroying steroids provides the enhanced effectiveness with respect to accumulation of intermediate steroid compounds. The preferable product of steroid accumulation if 9α-hydroxy-4-androstene-3,17-dione.

EFFECT: improved method for construction of strain.

8 cl, 5 dwg, 7 ex

FIELD: molecular biology, biochemistry, microbiology, medicine.

SUBSTANCE: method involves the simultaneous amplification of DNA and RNA targets and E. coli plasmid fragment or E. coli 16S-RNA as universal standard. For amplification two pairs of primers with the identical annealing temperature are used being primers of the first pair are specific to the target nucleotide sequence and primers of the second pairs of primers - to nucleotide sequence of the internal standard. A number of target copies are determined by data of electrophoretic separation of target amplicons and the universal internal standard. Using the invention allows carrying out the determination of amount of copies of DNA and RNA targets.

EFFECT: improved assay method.

4 dwg, 1 tbl

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, 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

FIELD: biotechnology, in particular isolated DNA molecule providing plant disease resistance and method for providing of disease resistance to plants.

SUBSTANCE: DNA molecular containing N1M1 is isolated. Recombinant vector including active in plant promoter functionally bonded with said DNA is constructed and plant is transformed by this vector.

EFFECT: decreased technological charges and increased land productivity.

9 cl, 37 dwg, 18 tbl, 10 ex

FIELD: molecular biology, biochemistry, medicine, oncology.

SUBSTANCE: invention relates to DNA sequences found in analysis of mDNA from squamous carcinoma cellular lines of different origin wherein these DNA sequences represent transcripts from rearranged genes SCCA1 and SCCA2. Result of rearrangement is formation of fused gene consisting of exon 2-7 of gene SCCA1 and exon 8 of gene SCCA2, or exons 2-7 of gene SCCA2 and exon 8 of gene SCCA1. Prepared expressing vectors comprising above said combinations of exons of two genes provide synthesis of corresponding fused protein in host-cell. Proposed sequences of nucleic acids and genetic constructions based on thereof represent novel agents for diagnosis squamous carcinomas.

EFFECT: valuable biological and medicinal properties of transcripts.

8 cl, 9 dwg, 1 tbl, 5 ex

FIELD: gene engineering.

SUBSTANCE: invention relates to method for modification target endogenic gene or chromosomal locus in eucaryotic cells. Claimed method includes production of large cloned genomic fragment having more than 20000 n.p. and designing based on the same large targeting vector (LTVEC) by using bacterial homological recombination. Further LTVEC is introduced into eucaryotic cells to modify endogenic gene or chromosomal locus. Finally assay is carried out to determine of allele modification in such cells. Also disclosed is application said cells for generation of organisms carrying such genetic modification.

EFFECT: method for modification with large DNA sequences.

26 cl, 6 dwg, 2 tbl, 5 ex

FIELD: medicine, immunology.

SUBSTANCE: the present innovation deals with specific prophylaxis of smallpox and viral hepatitis B. The kit contains two tablets each contains stabilizing additives, a filler and lyophilized alive viral material worked out based upon recombinant VOV strain at typical VOV properties expressing proteins preS2-S and HBs virus of hepatitis B virus, the first immunizing dosage corresponds to minimal quantity of viral material being sufficient to obtain weak immune response in the body in case of insignificant at insignificant reactogenicity, and immunizing dosage of the second - maximal quantity of viral material that causes pronounced and prolong immune response in the body at no negative side action. The technique of applying the kit of bivaccine tablets, first, one should use the 1st tablet at minimal dosage of bivaccine, as for the 2nd tablet - with maximal dosage of bivaccine it should be taken till the moment of developing humoral answer (in 7-14 d) after injecting the 1st tablet at minimal immunizing dosage of bivaccine. The innovation enables to create stable immunity.

EFFECT: higher efficiency.

4 cl, 5 ex, 6 tbl

FIELD: biotechnology, medicine, immunology.

SUBSTANCE: invention relates to recombinant vaccine against HIV-1 that represents a combination of immunogens as micelle-like particles comprising recombinant plasmid DNA pcDNA-TCI covered by conjugate spermidine-polyglucine with recombinant protein TBI comprising conservative T- and B-cellular virus-neutralizing epitopes in the stabilizing system. In immunization using this created vaccine production of specific antibodies possessing the virus-neutralizing activity is induced as well specific cytotoxic immune response also.

EFFECT: valuable medicinal properties of vaccine.

1 tbl, 5 dwg, 5 ex

FIELD: biotechnology, genetic engineering.

SUBSTANCE: invention describes recombinant plasmid DNAs constructed in vitro that comprise artificial genes for light and heavy chains of full-scale human antibody prepared by genetic engineering methods. These genes are created on basis of variable fragments of light and heavy chains of recombinant antibody 1F4 and constant human genes IgG1, cytomegalovirus promoter and polyadenylation site BGH. Plasmids provide biosynthesis of recombinant full-scale human antibodies of class IgG1 in mammalian cells. These antibodies interact specifically with smallpox vaccine virus. The affinity constant for prepared recombinant antibodies is 3.54 x 109 ± 0.38 x 109 M-1. Plasmids are used by combined transfection of human cells HEK 293T. Prepared full-scale recombinant antibody against protein of size 27 kDa of smallpox virus vaccine can be used as a base for creature of pharmaceutical preparations used for diagnosis of some post-vaccine complications caused by smallpox virus vaccine. Also, preparations will comprise decreased therapeutic doses of immunoglobulins that will provide minimal undesirable immune response in patients after administration of the preparation.

EFFECT: valuable medicinal properties of plasmid DNA.

4 cl, 7 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention concerns genetic engineering, biotechnology, immunology and medicine and can be applied in obtaining antitumoral vaccine and melanoma treatment. Vaccine compositions are based on heat shock proteins and antigen peptides for treatment of tumor disease, the said compositions containing hybrid protein consisting of heat shock HSP70 family protein and one of specific melanoma MAGE peptides (A1, A2, A3) or gp 100, as well as suitable pharmaceutical carrier. The invention also claims a method for obtaining the said hybrid proteins by gene expression in synthetic recombinant vectors in producer strains of E coli BL21 (DE3) cells.

EFFECT: improved immunising power.

3 cl, 4 ex, 1 tbl, 2 dwg

FIELD: chemistry, biotechnology.

SUBSTANCE: invention relates to field of biotechnology and concerns obtaining factor VII protein by method of recombinant DNA. Recombinant plasmid DNA was constructed for expression of blood clotting factor VII in mammalian cells, which is product of ligating of fragment of cDNA of human factor VII gene, flanked by sites of restrictases Xhol and BamHI recognising, with large XhoI/BglII fragment of vector pEFZeo, including genes of resistance to ampicillin and zeocin. As result of BHK cell transformation with new recombinant plasmid, cell line BHK/F7 was obtained, which produces recombinant protein of factor VII with output of up to 40 mkg/ml.

EFFECT: obtaining cell line producing recombinant protein of factor VII.

2 cl, 4 dwg, 4 ex

Up!