SU1751207A1 - Recombinant plasmid dna ps t20, encoding human alpha- interferon in tobacco plants - Google Patents

Abstract

Use: for biotechnological production of interferon. Summary of the Invention: Cloning a human recombinant alpha interferon gene in an expression vector under the control of a constitutive 35S promoter and cauliflower mosaic virus, transferring the recombinant plasmid into Agrobacterium tumefaciens cells carrying a disarmed Ti plasmid, integrating both plasmids, transforming with leaf sheets and regeneration of high-grade transgenic plants producing recombinant human alpha interferon

Description

The invention relates to genetic engineering and can be used in the biotechnological production of interferon.

Methods are known for producing human interferon-producing cells, bacterium, yeast.

A method for the introduction and transient expression of the human interferon gene in turnip plants as part of a cauliflower mosaic virus is described. However, this method does not allow for the integration and stable maintenance of the interferon gene in the plant genome, which is due to the use of a virus as a vector. Stable insertion and expression of the interferon gene can be accomplished only by genetic transformation of plants, as has been shown for a number of other genes.

The aim of the invention is the creation of a plasmid encoding human alpha interferon in transgenic plants.

The advantages of transgenic plants over other organisms - interferon producers - are the absence of the need to use for the production of large quantities of plant biomass of expensive specialized equipment and premises and growth media, sterile growing conditions, highly qualified personnel.

The invention includes the cloning of the recombinant human alpha interferon gene into the pST6 expression vector under the control of a strong constitutive 35S promoter from the cauliflower mosaic virus, transferring the resulting recombinant plasmid pST20 into the Agrobacterium tumefaciens C158 strain, carrying the unarmed pGV2222 plasmid disarmed with the Ti plasmid, which is unarmed. , genetic transformation by the method of leaf disks of Nicotiana tabacum cultivar Samsun obtained by the strain of agrobacteria and regeneration on the selective medium of high-grade transgenic

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plants producing recombinant human alpha interferon.

Example. Cells of E. coli K12 strains containing plasmids pINF, pUC19 and plink (pBR322 with a polylinker from pUC19 embedded with EcoRI and Hind 111 sites) were grown on a full nutrient medium with antibiotics for 12 hours at 37 ° C and plasmid DNA was isolated from Hi / ft by the alkaline method, which is then purified by gel filtration on a mini-column with arose 2B. 5 μg of plasmid pINF DNA was digested with restriction enzymes EcoRI (25 units) and Pstl (25 units) for 1 hour at 37 ° C. 2 μg of plasmid pUC19 DNA was digested with restriction enzymes pst I (10 units) and Hfndlll (10 units) 1 hour at 37 ° C. 5 µg of plasmid pllnk DNA was digested with EcoRI restriction enzymes (25 units), Hindlll (25 units) and Pvul (25 units,) for 1 hour at 37 ° C, then gel-filtered on a mini-column with sepharose 2B to separate small EcoRI-Hindlll fragment (psGLSHKy r from pUC19) from the remaining fragments of the plasmid. Together, samples containing the small EcoRI-Hindlll DNA fragment of the plasmid plink and the digested EcoRI and Pstl DNA of the plasmid pINF, and 0.1 µg of the plasmid pUC19 digested with Pstl and Hindlll are added to them, the mixture is deproteinized with chloroform and precipitated with ethanol for 12 hours at -20 ° C. The precipitate is dissolved in 10 µl of TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA), 1/5 part is taken and ligated with 2.5 units of DN4 T4 ligase in 50 µl of ligase buffer 12 h WITH.

The ligated DNA is transformed into E. coli TGI competent cells and plated on an ENDO selective medium, which allows the presence of DNA insertion in the pUC19 plaomid to be determined by colony coloration and also containing ampicillin (100 mg / l) White colonies grown at 48 ° C, The share of which is approximately 30% of the total number of ampicillin-resistant transforms, is purified on the same medium to individual colonies, and plasmid DNA is isolated from several cells, which is then digested with EcoRI, BamHl, Pstl and Hindlll restrictases. After analysis of the restriction DNA in an agarose gel, variants were selected from which the inf gene was inserted between two BamH1 sites.

In the next step, plasmid DNA was preparatively isolated from the isolated clone of bacteria, as well as from E. coliTGI cells containing the rZTb expression vector, as indicated. 5 μg of the plasmid pUC19 with the inserted interferon gene and 2 μg of the DNA of the plasmid pST6 are digested with the restriction enzyme BamHl (50 units) and the whole DNA is mixed

pUC19: inf plasmids with 0.2 µg of pST6 plasmid, chloroform and precipitate with ethanol and dissolve the precipitate in 10 µl of TE buffer. 1/5 part of the mixture

DNA is ligated in 10 μl of ligase buffer with 2.5 units. DNA ligase T4 18 h at 4 ° C. The ligated DNA transforms the competent E. coli TGI cells and selects the transformants on a nutrient medium from 100

0 µg / ml ampicillin. Clones grown at 37 ° C for 16 hours were purified on the same medium and plasmid DNA was isolated from them, which was digested with restriction enzymes EcoRI, Pvull, Bglll and BamHl and the clones were selected.

5 of which the interferon gene is inserted into the rZTb vector in the desired orientation,

In the next step, the resulting recombinant plasmid pST20 is transferred from E.coll cells to the cells.

0 agrobacteria. To this end, bacteria A tumefaclens C158 rifR (pGV2260), E. coli TGI (pRK2013) and E. coli (pST20) are grown in nutrient broth with aeration to achieve a cell suspension density

5 108 cells / ml. Mix 0.1 ml of the cell suspension of all three strains and apply it to a nitrocellulose filter, which is placed on a full nutrient medium for 18 hours at 30 ° C. Bacteria are washed off the filter and

0 are plated on selective medium containing ampicillin (100 μg / ml), rifampicin (100 μg / ml), with pectin and qing (100 μg / ml) and streptomycin (100 μg / ml). Colonies grown in 3 days on selective medium with

5 30 ° С is purified on the same medium to separate colonies and total DNA is extracted from cell biomass, 10 µg of DNA is digested with 20 units of BamHl restriction enzyme for 1 hour at 37 ° С, and fractionated by electrophoresis

0 0.7% agarose gel and transferred to a nitrocellulose filter by the Southern method. Hybridization of the DNA immobilized on the filter with the 32P-labeled DNA plasmid pST20 is carried out, the filter is washed off from he5, the bound tag is exposed and the filter is exposed for 24 hours with the X-ray film. On a radioautograph, DNA from the original Agrobacterium strain yields one hybridization zone with pST20, corresponding to the ampicillus susceptibility gene region on plasmid pGVC2260, and DNA from transconjugants clones gives 3 hybridization zones, which proves the integration of plasmids pST20 and pGV2250 in agrobacteria cells,

5 Agrobacterium strains obtained carrying the integrated plasmids pST20 and pGV2260 are used to infect tobacco leaf disks. Bacteria are grown on minimal medium A at 30 ° C and suspended in RMNO medium to density

10 cells / ml. The leaves of sterile tobacco plants of N. tabacum cultivar Samsun at the age of 3-4 weeks are immersed in bacteria suspension for 2-3 minutes, soaked with sterile filter paper and placed on RMNO medium, where they are incubated for 2 days in the dark at 25 ° C, after which the transfer t on medium for organogenesis (salt medium Murasigi and Skoog + 1.5 µg / ml BAP + 1 µg / ml of vitamin Bi + 0.8 µg / ml of vitamin Wb + 250 µg / ml claforan + 50 µg / ml kanami- tsina) and incubated in a camera with a photoperiod of 16/8 hours 3 weeks. The resulting shoots are transplanted onto a rooting medium that is different from the average food for organogenesis by replacing BAP with 1 µg / ml IAA, and incubate for another 3 weeks to accelerate the shoots.

Plants formed on kanamycin selective medium are tested for the presence of interferon gene DNA and its expression. The presence of DIC of the interferon gene in plants is determined by the dot-hybridization method, 30 µg of total DNA isolated from regenerated plants is denaturated and applied to a nitrocellulose filter, followed by hybridization of the DNA-labeled interferon gene on the filter ( small BamHI fragment of plasmid pST20) Plants that give a hybridization signal contain the DNA of a human interferon gene.

To establish the transcription of the interferon gene in transgenic plants, total RNA is isolated from the leaves, fractioned by electrophoresis on a 1.2% agarose gel under denaturing conditions with formaldehyde, transferred to a nitrocellulose filter, and hybridized with

P-labeled interferon gene DNA. As a standard, 0.1 ng of the DNA of the small WatN fragment of the pST20 plasmid, as well as 18S and 25S ribosomal RNA, are used. The conclusion about the transcription of the interferon gene in transgenic plants is made on the basis of the appearance of a hybridization zone corresponding to RNA molecules with a size of 0.95 kb.

The protein product of the interferon gene in transgenic plants is immunologically determined using monoclonal antibodies against human leukocyte interferon. 100 mg leaves are ground in a mortar and extracted with buffer (25 mM Tris-HCl, pH 8.35; 195 mM glycine) Cell fragments precipitated by centrifugation, ethanol is added to the supernatant to a final concentration of 20% and the solution is filtered through nitrocellulose filter. The filter is washed 4 times with 5 times

min with solution B (10 MM-NaH2P04 / Na2HP04 with 0.15 M NaCI; 0.3% Tween-20; pH 7.2) 5 μl of a solution containing monoclonal antibodies against human leukocyte interferon is applied to the filter surface and incubated for 12 hours at 4 ° WITH. The filter is washed 4 times for 5 minutes in solution B and 5 µl of a solution of labeled antibodies against mouse IgG is applied to it. Incubate for 30 min at 20 ° C and wash the antibodies on the filter 4 times for 5 min with solution B. Sv zavshies antibodies are visualized by illuminating the filter with long-wave UV light. 500 units are used as standard. human leukocyte interferon. The production of interferon protein by transgenic plants is evidenced by the appearance of strong fluorescent luminescence of preparations on filters.

RMNO medium: 1x macrosalt, 1x micro-salt, 1 mg / l Fe-chelate, 440 mg / l CaCIa, 100 mg / l-meso-inositol, 1 mg / l vitamin B1, 0.1 mg / l 2,4-D, 0 , 04 mg / l kinetin, 3 mg / l IAA, 3% sucrose, 8 g / l agar

Wednesday A: 10.5 g / l NROL. 4.5 g / l КН2Р04, 1 g / l (МНФЗО. 0.5 g / l sodium citrate -2Н20

Nutrient medium for bacteria: 10 g / l tryptone, 5 g / l yeast. extract, 10 g / l NaCI.

Agarized nutrient medium: m - septon agar (MPA) produced by IEM im. N.F. Gamalei

Claims (1)

Claims of the invention Recombinant pST20 plasmid DNA encoding human alpha interferon in tobacco plants, 7.7 kb in size, containing: -BamHI - BamHI DNA fragment of a PINF plasmid of 0.9 m in size in the human alpha interferon genome; -BamHI - BamHI DNA fragment of the rZTb vector of 6.8 mp in size; - restriction sites: two ECORI sites, one of which is located at a distance of 2.6 kb. from the first ECORI site; Three Pvull sites located at a distance of 0.05,1.45 and 2.95 kb. from the first ECORI site; - two Hindlll sites located at a distance of 2.15 and 3.75 kb. from the first ECORf site, - two BamHI sites located at a distance of 2.6 and 3.5 tons bp from the first ECORI site; - one 5t1 site located at a distance of 3.75 tons of the original, from the first ECORI site; -regulatory plots: P35S promoter - cauliflower mosaic virus promoter ;, PNOS promoter - nopaline synthase gene promoter from Ti plasmid; .- region m MOZ - transcription terminator and polyadenylation signal and RNA from the Z1 region of the nopeline synthase T gene - plasmid; - region 3 OCS - transcription terminator and polyadenylation signal and RNA from the 3 region of the octopinsyntase gene TI - plasmid; 0 -RR and LR regions - 25 bp right and left signal repeats flanking T-DNA; -genetic markers: - gene pr t P - gene for resistance to kanamycin in plants; the CPH gene is a gene for resistance to ampicillin in bacteria; The Sm gene is a gene for resistance to streptomycin in bacteria; Sp gene is a gene of resistance to spectinomycin in bacteria.