SU1436887A3 - Method of producing recombination plasmodium dna encoding growth hormone of rat or human being - Google Patents

Abstract

The invention relates to the field of genetic engineering and concerns a method for producing plasmid DNA encoding rat or human growth hormone. The method consists in isolating mRNA from the pituitary gland, doubling the obtained RNA, obtaining single-DNA by using reverse transcriptase, doubling the obtained cDNA, selecting the desired dDNNA fraction and cloning it in pBR 322, 2 table.

Description

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The invention relates to genetic engineering and concerns a method for producing a plasmid DNA encoding rat or human growth hormone.

The method consists in isolating mRNA from the pituitary gland, doubling the obtained RNA, obtaining single-DNA by using reverse transcriptase, doubling the obtained DNA, selecting the desired dDNIC fraction and cloning it into pBR 322.

Example 1. Cultured rat pituitary cells (a subclone of the GH-1 cell family), obtained from the American Type Culture Collection, are used as a source of mRNA for the synthesis of rat growth hormone. In these cells, if growth is carried out under normal conditions, the growth hormone mRNA is contained in small quantities, from 1 to 3% of the total RNA content having poly-A, However, the growth hormone mRNA content is significantly increased compared to other mRNA cells as a result of the stimulating effect of thyroid hormones and glucocorticoids. RNA was isolated from 5-10 cells grown in suspension, in which i iMonb deoxymethasone and 10 mmol β-trI1 odirone were added to collect growth hormone 4 days before collecting cells. Polyadenylated RNA is isolated from the cytoplasmic separating membranes of cultured cells by known methods.

Polyadenylated RNA is isolated by chromatographic analysis of the entire RNA preparation on oligo (dT) cellulose.

A reverse transcriptase, isolated from a virus, avian myeloblastosis, was obtained from Doc. D.J. Byrd, Life Science Inc., St. Petersburg, Florida; it is used to copy the entire polyadenylated RNA molecule obtained from cultured cells into cDNA. Reactions are carried out in a solution of 50 mmol Tris HCl, pH 8.3, 9 mmol. MgCl. 30 mmol of sodium chloride, 20 mmol of beta mercaptoethanol, 1 mmol of each of the three non-radioactive deoxyribonucleoside triphosphates, 250 mmol of the fourth deoxynucleoside triphosphate, labeled with 32P in alpha position, with a specific radioactivity of 50-200 Ci / mol 20 μG / ml of oligo d 12-18, obtained from the company (ollabora Research, Vaptman, Massachusetts, 100 mg / ml of polyadenylated RNA and 200 units / ml of reverse transcriptase. The mixture is incubated at A5 C for 50 min. After adding EDTA- Na, 25 mmol, the solution is extracted using phenol saturated with water, then The layer was subjected to chromatography on a Sephadex G-100, with a column with a diameter of 0.3 cm and a height of 10 cm, in a mixture of 10 mmol Tris-HC1, pH 9.0, 100 mmol sodium chloride, 2 mmol EDTA.Nucleic acid eluted in an empty volume, precipitated with ethanol after adding ammonium acetate to 0.25 m, pH 6.0. The precipitate is collected by centrifuging, the resulting tablet is dissolved in 50 ml of freshly prepared 0.1 M sodium hydroxide solution and incubated for 20 min to hydrolyze RNA. The mixture is neutralized with the addition of 3 N sodium acetate solution, pH 4.5, and the resulting 32 P-cDNA is precipitated with ethanol, and then redissolved in water. Separate portions of single-strand cDNA are analyzed on natural polyacrylamide gels. The gels are dried, labeled with 32P cDNA, analyzed by autoradiography using a Kodax No-Sereen-2T film (brand name, Eastian Kodak Corporation, Rochester, New York). After separation by electrophoresis on a gel, a weak absorption band is found, corresponding to DNA from about 800 bp.

The resulting single-strand cDNA is treated with reverse transcriptase to synthesize a complementary strand. The reaction mixture contains 50 mmol Tris-HCl, pH 8.3, 9 mmol MgCl, 10 mmol dithiothreitol, 50 mmol three unlabeled deoxy ribonucleoside triphosphates, 1 mmol of labeled 32P in the position of alpha nucleoside triphosphate with a specific radioactivity of 1-10 Chi / mmol 50 MCH / ML cDNA and 220 units / ml of reverse transcriptase. The reaction mixture is incubated at 45 ° C. for 120 minutes. The reaction is interrupted by adding EDTA-Na to 25 mmol, extracted with phenol and subjected to chromatographic analysis on Sephadex G-lOO, and then precipitated with ethanol. Portions of the product reac.1.1 and with a rate from 500 counts per minute to 100 counts per minute are analyzed using

gel electrophoresis. By comparing with standard samples, it is established that the absorption band of the heterodisperse is concentrated around 800 nucleotides in length.

Treatment of the complete cDNA, on which the rat pituitary cell mRNH structure is copied, with the Hha I endonuclease leads to the formation of two main DNA fragments, one containing approximately 320 nucleotides (fragment A) and the second 240 nucleotides (fragment B). The analysis of the nucleotide sequences of fragments A and B is carried out as described above by Mahat and Gilbert, which confirms the following fact: these fragments are in fact those parts that contain the genetic code encoding the synthesis of rat growth hormone.

In cleavage of DNA containing 800 bp. with the Kha T endonuclease, among the major dissection products, two fragments are found, corresponding in length to fragments A and B.

Since the rat growth hormone synthesis cDNA, containing approximately 800 bp, is purified before being treated with Hha I, the DNA must be processed to remove all unconnected ends. Under actual conditions, the treatment was carried out in order to remove such unpaired KOHI; OB was carried out before the separation step on electrophoresis in a mixture containing 25 µl of 60 mmol Tris-HCl, pH 7.5, 8 mmol magnesium chloride, 10 mmol beta-mercaptoethanol, 1 mmol of ATP and 200 mmol of each of aATP, dUTO, dGTF and dTTO. The mixture is incubated with polymerase I DNA (1 unit) isolated from E. coli strain at 0 ° C for 10 min in order to remove all protruding 3 ends in an exonucleolytic method and fill all 5 protruding ends. Polymerase I DNA is produced by Boehringer-Mannheim Biokemikalz, Indianapolis, Indiana.

The cDNA, which controls the synthesis of rat growth hormone and contains approximately 800 bp, is processed at

process 30 pieces. nuclease SI having an activity of 1200 units / ml, which was obtained from Myles Laboratories, Elkhart. Indiana, in a mixture containing 0.03 mmol sodium acetate, pH 4.6, 0.3 mmol sodium chloride,

4.5 mmol zinc chloride, at 22 C for 30 min, and then incubated for an additional 15 min at. In order to terminate the enzyme reaction, Tris-base with a final concentration of 0.1 mmol EDTA to 25 cmol and tDNA is added to the mixture.

After ethanol extraction of the reaction mixture and chromatographic analysis on Sephadex G-100, 32 p-cDNA, eluted into the empty volume, precipitated with ethanol. As a result of this treatment, a high yield of cDNA molecules is provided, the ends of which are connected by a base. The Hind III linker is ligated with cDNA by incubation at 14 ° C in a mixture of 66 mmol Tris-HC1, pH 7.6,

6.6 mmol magnesium chloride, 1 mmol ATP, 10 mmol dithiotreitol, 3 mmol Hind ITT decamer with a reading of 10 counts per minute / mol and T4 DNA ligase,

approximately 500 units / ml for 1 hour. To deactivate the ligase, the reaction mixture is heated to 65 ° C, which is maintained for 5 minutes. Preliminary, KC1 with a final concentration of 50 mmol, beta-mercaptoethanol with a final concentration of 1 mmol and EDTA with a final concentration are added to enzymes containing 1CIM 150 units / ml Hsu I or Hind III.

0.1 mmol. Mixture No. is kept for 2 hours at 37 ° C. The Hind III and Hal III endonucleases are manufactured by New England Bio-Dabo, Beverly, Massachuset. The reaction product is analyzed by gel electrophoresis according to an example; a peak is found corresponding to a sequence of approximately 450 nucleotides, along with individual fragments of dissected Hind III decamer. Plasmid pBR-322, containing the gene, controlling ampicillin resistance and the only recognition site for the Hind TSH enzyme,

the power of adding chemically synthesized 55 inside the aforementioned Hind III gene linker is dissected at the site of recognition

The resulting, as a result of the reaction for Hind III with the endonuclease Hsu I, the product whose molecule consists of themes is treated with alkaline phosphate of two strands, with a concentration of 2-5 μg / ml of the EAPF type, Worthington Biocem.

Corporation, Freehold, New Jersey. The enzyme is in the reaction mixture at a concentration of 0.1 units. The microgram DNA reaction mixture was incubated in 25 mmol Tris-HC1 at pH 8 for 30 minutes at 65 ° C, then extracted in phenol to remove phosphatase. After phosphatase treatment of the plasmid DNA, Hind III is cut with the endonuclease and ligiru ends of the molecule} in a molar ratio of 3 mol of the plasmid per I mol of cDNA. The mixture is incubated in a mixture of 66 mmol Tris-HCl, pH 7.6, 6.6 mmol magnesium chloride, 10 mmol Dithotraitol and i mmol ATP for 1 h with 50 units / ml DNA ligase T4.

The mixture, in which the ligation proceeds, is added directly to the human oral hypophysis, quickly filling the E. coli X-1776 strain. At the same time, the cells are expanded to a specific density of approximately 210 cells / ml in 50 ml of medium containing: tryptone 10 g / l; yeast extract 5 g / l, sodium chloride 0 g / l, sodium hydroxide 2 mmol; diaminopimellic acid 100 µg / mp, thymine 40 µg / kp at 37 C. Cells were collected using a centrifuge for 5 minutes at a speed of 5000 Gt at 5 ° C, resuspended in 20 ml of cold sodium chloride solution, 1 O mmol centrifuged re-suspended in 20 ml of transformation buffer containing 75 mmol of CEiCl, 140 mmol of sodium chloride and 10 mmol of Tris-IS1, pH 7.5, then the mixture is kept for 5 minutes on ice. Next, the cells are centrifuged and resuspended. transformation buffer 0.5 ml. The transformation was carried out with the displacement of 100 µl of the cell suspension with 50 µm of DNA recombinant (1 g / ml). The mixture is incubated for 15 minutes, then at 0 ° C.

4 minutes at 29 ° C and, finally, for 30 minutes again at 0 ° C. Next, the cells are transferred to plant agar for growth under selective conditions,

Recombinant colonies are selected by growing on a nutrient medium containing ampicillin. If no growth is detected, then on a nutrient medium containing tetracycline with a concentration of 20 g / mp. Ten such cell colonies were obtained, each of which contains a plasmid

with about 800 bp, the molecule of which breaks under the influence of the enzyme Hind TIT.

A rat growth hormone DNA containing 800 bp was prepared in preparative amounts from a recombinant pGRK-1 clone and its nucleotide sequence was analyzed.

The sequence of mRIC derived from the genetic sequence is presented in Table. one.

The release of mGNA, human growth hormone growth hormone is carried out as in example 1} only in this case, the biological source of the material is human pituitary tissue. Five surgical-freeze-dried in liquid nitrogen, after surgical removal, weighing between 0.4 and 1.5 g each, are thawed and finely ground in a 4 M rat guanidine thiocyanate solution containing 1 M mercapto ethanol buffer, pH 5.0, at 4 ° WITH. The homogenate layer on 1.2 ml of a 5.7 molar solution of cesium chloride containing 100 mmol

30 EDTA and centrifuged for 18 hours at a speed of 37,000 rpm on a Beckmann ultracentrifuge with a rotor of the SW 50.1 type (Beckmann Company Instrument, Fullerton, California). Wherein

35 RNA is collected at the bottom of the tube. Next, the purification was carried out using a column on oligo-dT and precipitation with a sucrose gradient solution in accordance with the example. About

40 10% of the RNA thus isolated contains the genetic code for the synthesis of growth hormone; this fact is established by the introduction of a radioactive precursor compound containing

45 amino acids, in a hormone that inhibits growth, which precipitates in a system of cells in which translation did not occur, obtained from the AVIER embryo.

50

The production of human growth hormone cDNA is carried out in accordance with Example 1. Growth hormone cDNA is fractionated by electrophoresis on a gel and the material that is moved to a position corresponding to approximately 800 nucleotides in length is chosen for propagation. The selected fraction is treated with polymer oh I DNA according to 11D36887

Example 1 is then processed to combine the ends with the Hind TII linker. Next, the cDNA is recombined with the plasmid pBR-322, which is pretreated with alkaline phosphatase in the presence of DNA ligase. E, coli X-1776 is transformed with recombinant DNA, and then the strain containing the growth hormone DNA is extracted. A strain containing growth hormone DNA is grown in preparative amounts, growth hormone DNA is extracted from it, and then the nucleotide sequence is determined. It was installed

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It is believed that the isolated DNA of growth hormone contains nucleotides containing information for the synthesis of the complete amino acid sequence of human growth hormone. The first 23 amino acid-treated Hind III and alkaline phosphate methods for producing recombinant plasmid DNA encoding rat or human growth hormone, including incubating pituitary cells in the presence of 1 mmol deoxymethasone and 10 mmol 1-triiodothyron, separating mRNA from the cytoplasmic membrane fraction of the hypophysis cells, and cDNA synthesis using reverse transcriptase, incubating the mixture of cDNA with DNA polymerase in the presence of dATO, aGTP, acTP and aTPT at pH 7.5 1 & 10 C, the allocation of the cdnDNA fraction, which has 800 bp, the addition of Hind III linkers to the selected dsDNA, the displacement of the DnDNA from DNA obtained from the pBR 322 plasmin, previously

Fatase at pH 8.0 and for

You have the growth hormone: H N-FE-Pro-Tr-1le-Pro-Leu-Ser-Arg-Leu-Fe-Asp-Ask-Ala-Met-Leu-Arg-Ala-His-Arg-Leu-His -Glto-leu. The rest of the sequence is presented in table. 2,

Claims (1)

Hind III and alkaline phosphate A method for producing recombinant plasmid DNA encoding rat or human growth hormone, including incubating pituitary cells in the presence of 1 mmol deoxymethasone and 10 mmol 1-triiodothyron, separating mRNA from the cytoplasmic membrane fraction of pituitary cells, purifying and synthesizing cDNA using reverse transcriptase, incubating the mixture with DNA polymerase in the presence of dATO, aGTP, acTP and aTPT at pH 7.5 and 10 C, the distribution of the cdna DNA fraction, which has 800 bp, the addition of Hind III linkers to the selected DNA, DNA displacement from plasbid pBR 322, previously about 20 worked with Hind III and alkaline phosphatase at pH 8.0 and for 30 min followed by incubation mixture for 1 h at 1A and pH 7.6  in the presence of ATP and DNA ligase and extraction of the obtained recombinant DNA. in tf