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.


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.


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