SU1707078A1 - Recombinant plasmid dna pthy314, encoding polypeptide with properties of human @@@-thymosine, recombinant plasmid dna pthy12 - an intermediate product for its construction, and the strain of bacteria escherichia coli - a producer of polypeptide with properties of human @@@-thymosine - Google Patents

Abstract

This invention relates to biotechnology, in particular to genetic engineering. The aim of the invention is to increase the level of synthesis of a polypeptide with the properties of human thymosin ai. A recombinant plasmid pTNU314 was obtained that encodes a peptide with human thymosin properties, E. coll VKPM B-5056, providing synthesis of a polypeptide with thi properties. The invention relates to biotechnology, in particular to genetic engineering. Thymosin is one of more than 30 peptides of low mol.m. (1000-15000). secreted from the thymus gland (thymus gland), has a powerful general immunostimulating activity. Thymosin "1 is synthesized in the body as a precursor, human mozine, and recombinant plasmid DNA pTH12, which is an intermediate product for the production of the plasmid pTHU314. The recombinant pTNU12 plasmid contains a synthetic polypeptide gene with human thymosin a properties, consists of the Hind Ill / Cla I - DNA fragment of the plasmid pCPG2, containing the tandem of the A2 and A3 transcription promoters of the Tachophagous region. synthetic site of translation initiation, genes / -laktamazy and chloroamphenicol-acetyltransfergzy and phage transcription terminator). Clal / Htnd II - Fragment containing the systhetic gene. coding c: mu: th peg - tidtimosin th person. Rexmn-mZg -n plasmid DNA pTNUZ 4 contains a polypeptide gene with human tyrosine A properties, which constitutively expresses in E. coli cells as part of PSPU chimerae. Tc-necrosis factor; timazgn ;;. The Escherichia coli SG20050 / pTHV314 strain provides the biosynthesis level of human thymosin thymus peptide up to 8–10 μg / ml with a simplified technology ;. his highlighting. 3 sec. f-ly. from which it is cleaved and then subjected to N-terminal acetylation. N-desacetylthymosin sp has a set of biological activities on the peptide, in particular, thymosin stimulates T cells, inducing the synthesis of interpeykin-2 and / -interferon, making them effective against a wide range of pathologies such as С Ssl I and

Description

infectious and neoplastic diseases, rheumatism, acquired immunodeficiency syndrome (AIDS), etc. The combined use of thymosin M in medical applications with other drugs, such as aspirin, is promising in immunotherapy of lung cancer. The drug aspirin-timosin OH prevents infection with the AIDS virus. The closest technical solution to the present invention is the plasmid on pTH pp / gal DNA encoding peptide synthesis with the properties of human thymosin a 1.

The known plasmid has a number of disadvantages, among which is the low expression level of the fusion protein (less than 1%), the need to induce protein biosynthesis, which complicates the technology of cultivation of bacterial cells, the fusion analogue protein contains 23 methionine residues. which makes the recovery process of the target product time consuming.

The aim of the invention is to increase the level of synthesis of a polypeptide with human thymosin n properties.

This was designed by recombinants of the plasmid pTH12, which is intermediate to construct a recombinant plasmid pTU314, which encodes a constitutive synthesis of a hybrid protein, from which the peptide with human thymosinate properties is cleaved by hydrolysis with bromine. The obtained strain E. coli SG 20050 / pTNU314. providing high vposenb expression of the hybrid protein - tumor necrosis factor - it is thymosin, from which 8-10 μg of timosin a is obtained per 1 ml of cell suspension at a cell density of 10 cells / ml.

Recombinant plasmid DNA pTNU314, which encodes a chimeric protein - tumor necrosis factor - thymosin a. characterized by mol. 2.57 gid (3.98 kb.) And referring to:

Pstl-Sau3AI - DNA fragment of plasmid pTNF314 D. containing the tandem transcription promoters A2 and A3 of the early T7 bacteriophage region, semisynthetic gene for human tumor necrosis factor with a synthetic translational initiation region and part of the gene / H-lactamase (1.48 t.po.); Clal / Pstl - fon1menta | DNA plasmid pTN12 containing a synthetic gene. Thymosin 7i coding thymic peptide, chloramphenicol acetyltransferase gene. phage I transcription terminator and part of the gene / -lactameae (2 49 kb): Sau3A / Clal, a synthetic linker that connects the tumor necrosis factor and thymosin zi genes (15n.o.) in a single reading frame.

Recombinant plasmid DNA pTNU314 as genetic markers

contains genes of / 3-lactamase and chloramphenicol acetyltransferase, which determine the resistance of E. coli cells transformed with the plasmid pTHU314 to chloramphenicol and penicillin antibiotics, as well as unique restriction sites. located at the following distances to the right of the Kpnl site: BstEII — 405 nucleotides; Bglll - 492 nucleotides; Hindlll - 586 nucleotides. Pstl - 3779 nucleotides.

Recombinant plasmid DNA pTHI12, which is an intermediate product in the construction of recombinant plasmid DNA pTU314, is characterized by mol.m. 2.58 MD (4.02 kb)

and consists of:

Hlndlll / Clal - DNA fragment of the plasmid pCPG2 containing the tandem of the A2 and A3 transcription promoters of the early T7 bacteriophage region, the synthetic translation initiation region, the / lactamase and chloramphenicol acetyltransferase genes and the phage A transcription terminator (2.49 kb. ): Clal / Hindlll - fragment containing a synthetic gene encoding thymic peptide thymosin d (0.1 kb).

Recombinant plasmid DNA pTHI12 contains genes / β-lactamase and chloramphenicacetyltransferei as genetic markers, which determine the stability of the plemide-transformed pTHI12 E.coli cells to penicillin antibiotics and chloramphenicol, as the protagonist, aphidnn, asparating

BamHI site: Clal - 24 nucleotides; Bglll - 75 nucleotides; Hindlll - 118 nucleotides: Ncol - 729 nucleotides: Pst - 2573 nucleotides

In order to obtain an artificial gene coding for human thymosin, phosphoramidite solid phase method is used to synthesize eight oligodeoxyribonucleotides ranging from 21 to 29 nucleotide units. Ligase crosslinks are carried out in two stages.

Segment A:

IG III AATTCATCtA TATGAGCSATCCTCCTC ACACrrCTTCTCAAATCACC ACCA

(GTASSTATSTSTSTASSAAASLTSTSTALASALSMGGTTASTSTSTLSSGGT

four n f

Segment B:

tt g ui

STAAASATSTTALASAAAAAAAASAOSKTKSSAAASSTSAAASTA

V.AATTTCI 11ILI 11CTCCAACACCTXXTTCGACTTTTCATTOCA

tV vni

At the first stage, two four-component cross-links are performed, all oligonucleotides are phosphorylated before cross-linking using ATP and T4 polynucleotide kinase, with the exception of the 5-terminal oligonucleotide (I) from segment A and oligonucleotide III from segment B. On In the second stage, T4 DNA ligase 1 is stitched with A + B segments, which are pre-purified by electrophoresis in 15% PAAG containing 7 M urea. The resulting 101-stranded double-stranded DNA is purified by electrophoresis in 8% PAAG.

After that, the artificial gene of human timosin Gfi is cloned in the plasmid pGEM4. For this, the plasmid pGEM4 DNA was started digested with restriction enzymes EcoRI and Hlndlll, then the resulting vector was ligated with synthetic DNA. After transformation with a ligase mixture of competent cells of E.coll HB101, screening of the desired clones is carried out by in situ hybridization of colonies with (P) - labeled oligonucleotides (1) and (VIII). Plasmid DNA pTHI1-4 was isolated from clones hybridizing with both radioactive probes, analyzed using restriction enzymes Haelll and Mspl, and the structure of the insert was confirmed by determining the nucleotide sequence using the modified Maxam-Gilbert method.

The resulting recombinant plasmid DNA p T NU 1-4 contains the artificial thymosin fli gene. which can be transcribed by SP-6 RNA polymerase, and can be used for preparative production of RNA for cell-free translation using rabbit reticulocyte lysitants or wheat germ extracts.

After that, the synthetic thymosin sp gene is cloned according to the Clal and Hindlll sites into the plasmid pCPG2. The result is a new recombinant plasmid pTHI12, which is intermediate for constructing the target plasmid pTHU314, containing the thymosin p gene, preceded by the sequence of the translation initiation site (sequence Shine Dalgarno), and the transcription of thymosin a - specific RNA is provided with a tandem of strong promotions, and has been given a tandem of active promos T7. Thus, the pTH12 plasmid controls the direct expression of the thymosin ai gene and can be used for the cell-free peptide biosynthesis using the conjugate prokaryotic transcription-translation system.

For further construction. The plasmid pTHNF3l LD is used; it is a derivative of the recombinant plasmid pTNF311 Dc unchanged from the site-directed tumor gene C-terminal part of the human tumor factor factor gene. As a result, the ILeiss mutagenesis was replaced by the Leu residue, and the unique BamHI restriction enzyme site was introduced into the tumor necrosis factor gene. Plasmid pTNF314 D DNA is subjected to yy to local hydrolysis with the restriction enzymes BamHl and Pstl and the vector fragment is isolated

The plasmid pTH12 DNA is hydrolyzed with Clal and Pstl endonucleases, after which a 2.5-kb fragment containing the structural gene thymosin ai is isolated by electrophoresis in 5% PAAG. After that, the vector is ligated with the Clal - Pstl - fragment of the plasmid pTHU14 in the presence of a synthetic duplex:

GATCATTGCCCTGAT (IX)

TAACGGGACTAGC (X)

A portion of the ligase mixture is used for

transformation of competent cells of E.co 1

HB101. Transformants are plated on an agarized medium containing ampicillin.

and chloroamphenicol (50 µg / ml) and carried out

colony hybridization screening with P-ve fj

oligonucleotide GATCATTGCCCTGAT.

From the hybridizing clones,

pTNU314 plasmid DNA and analysis; yut

her with the help of restriction enzymes Haelii and Mep;

To obtain a producer strain

a peptide with a biological act IENSTUM of human thymosin d plasmid pTU31ch

transform competent cells

E. coli SG20050.

The resulting E. coli SG20050 / / pTNU314 strain is characterized by the following features.

Morphological signs.

Cells are small, thickened, visible, gram-negative. ceaseless.

Cultural features.

Cells grow well on simple nutrient media. When grown on Difco agar, the colonies are round, smooth, pressed, dull, shiny gray, the margin is even. With the growth in liquid medium (on the minimal medium with glucose or LB-broth) they form an intense even dregs.

Physical and biological signs. Cells grow at a temperature of from 4 to 40 ° C with a pH optimum of 6.8 to 7.0. As the nitrogen source, both the mineral salts in ammonium form and organic compounds in the form of peptone are used.

tryptone, gallstone from extract a and acid, etc. Amino acids, glycerin carbohydrates are used as a source of ul-type.

Antibiotic resistance

Cells exhibit resistance to ampicillin (up to 300 µg / ml) and Chlorminphenicol (up to 500 µg / ml). due to the presence of ppasmida. as well as tetracycline (up to 25 µg / ml). due to the presence of a vehicle.

The strain E.coll SG 20050 / pTNU314 determines the constitutive synthesis of a hybrid polypeptide - tumor necrosis factor - thymosin a. from which a peptide with the properties of thymosin a-human can be bit-, cleaved by the action of bromine cyan. when UPM is the level of hybrid protein biosynthesis (. it is over 20% of the total cellular protein of bacteria, which corresponds to 8 10 mm heptide per 1 ml of cell suspension.

The strain was deposited in the All-Union Collection of Industrial Microsurgery VNIIengtnka under the number VK VK PM-5056.

PRI me R 1 Khimik -fe omeme h t these p. h and and synthesis of not l g i blue structural gene

g imo / s -: h

Sich ee Oh and sleep leotidvig. tweacc essic phosphide-imidite method

. ..:. INTR / -JT OP9 C H R 11; AND 3 -H: M R.PI: p-dis

about

 R nf NII OT

: gs - nomkz protected osp fa m ,,: it about v - s - citritn., - scyl-2-deoxynuo -: - o / id-3 -C (met oxide and isoprogg. gmmino) -Fssfito8. L to t, in and ovova n-yl tetrazzhochom C g-: mes does a scale of 0.5–7 µmol. i-cgp. as a: t.pe iositega porous stek (500 A. pore size 4Q-8C microns1 i to which the first nucleosity of the DNA is attached via a 3-succinet bond (load 20 - J. O fi / g Bake; coziness; an intestinal g g of which, after the condensation reaction of the gc, went to rinse with -Pb mixture 0 tetra (id- g furan - pyridine-water (53: 2). After the end of the synthesis, the protective groups are removed by sequential treatment - lt t riet and l at-1 mo HI, a and concentrated ammonia In this case, the oligonucleotide is separated from the carrier 5- Limethoxytrityl group y is removed by acid treatment v oligonucleotide was purified by electrophoresis in 20% polyacrylamide gel. Yield containing 7M urea 1-5 pu

A mixture of 250 pmol of the oligonucleotide (fl) and 200 pmol of each phosphoryl-mediated oligochucleotide (Hi and (14) and 250 pmol of phosphorus) or the oligonucleotide (f)

warm 10 meme at 9 ° C in 100 μl of buffer. content of about 20 mM t rice HCI, pH 7.5. H) mM MgCli is then mrdte but cooled to 15 ° C with an excess of Al t p Al p to 0.2 mM.

5 digiotreitis up to 10 mM KOHUf 1 μm and 10 units. T4 DNA ligases The mixture is incubated for 6 hours at 15 ° C. then deproteinized twice phenol extraction. DNA is planted with ethanol and crosslinked products are isolated when

10 using electrophoresis in 15% PAAG containing 7M urea. Purified crosslinking product is eluted from the gel by electroelution onto DE-81 paper. which is washed several times with TE buffer (10 mM Tris-BUT;

15 pH 80; 0.5 mM EDTA) and ethanol. The nucleotide material is eluted with 1 5 M NaCI solution in TE-buffer and desalted on a Sephadex G-50 column. Segment A output 140 pmol

20 In a similar manner, segment B. Vyhod is obtained 180 pmol.

Next, 50 pmol of each A. segment are chemically administered. And B P 20 μl of buffer containing 20 mM trig-HCI, pN 7 5 Yu mM.

25 0.2 mg / gAT0. 1C mM dithiotreitis (buffer C) and 50 units of T4 LmK ligase. for 4 h at PPS,:, t cujHBiM v: deG1 | T h.textra 8. Mr. PAGE CER described Input

3 J PMOL STRUCTURE OF THE GPOMRZHE OF ETERNAL AND KO30 (-the end products of ligase reactions can be found in: a): t.J 3 n and: about the man d o b -, t - t t-o with t i.

 y and - f p 2 Recommended recombinant g pThy1-4

35Kl-tk, - akgeoi f. coli, JM101. containing. ch-lgg and pneg-4 DNA, grown in e-LP broth containing 100 m to g af-pitsiltin, dz stationary Phase, then the aunt is separated known

40 proaed- , si with modifications comprising what to the supernatant. sodium acid acetate after acidification. GNUA A is added to a concentration of 10 μg / m: the mixture is incubated for 20 minutes at

d5 37 ° C. extracted twice with a mixture of phenol-chloroform (1: 1) and the DNA is extracted with ethanol. The precipitate is dissolved in TE buffer containing 1 M NaCl. polyethylene glycol (PEG 6000) is added to

50 1.5%, incubated for 1 h at 0 ° C. centrifuged7 for 10 min at 10,000 rpm, and the precipitate is washed: watered with 70% ethanol. dried and dissolved in TE buffer; The yield of plasmid DNA was determined spectro55 photometrically at 260 nm using an extinction coefficient of 20 pu / mg.

DNA of plasmid PTNF314 D containing the mutant human tumor necrosis factor gene was isolated in a similar manner. as well as new recombination plasmid DNAs of rTNUG A. pTHV12 and p7HU314.

The resultant pressure is plasma. pGEM4 identical DNA was used to construct the plasmid pTH1-4. which is carried out as follows

5 μg of pGEM-1 plasmid DNA is incubated with EcoR and HindMI restriction endonucleases (20 units each) in 100 μl of buffer B (20 mM Tris-HCl: pH 7.5. 50 mM №Ci: 10 mM MgCl2: 5 mM mercaptoethanol) 1 h at 37 ° C. After incubation, the reaction is stopped by double extraction with phenol-chloroform (1: 1). Analysis of the completeness of hydrolysis and isolation of the vector EcoRI / Hindlll fragment was carried out by electrophoresis in 1% agarose gel. Next, 1 µg of the vector DNA is combined with 0.2 µg of the synthetic double-stranded fragment from Example 1 with 30 units. T4 DNA ligase in 50 μl buffer C for 6 s at 15 ° C.

A tenth of the reaction mixture was used to transform E. coli JM101 cells. for which cells are plated on agar. containing M9 medium, 0.2% glucose and 2 μg / ml thiamine. A single colony is added to 50 ml of nutrient broth and grown at 37 ° C to a turbidity of 0.3-0.5. Then the cells are cooled, precipitated by centrifugation (10 minutes, 5000 rpm), washed with a solution of 10 mM MgCl /. centrifuged, suspended in 20 ml of 0.1 M rasteorz CaCI: and incubated for 30 min at 0 ° C. After centrifugation, the cells are resuspended in 3 ml of 0.1 M CaCI and after 3 h, dt transformation is used. For this purpose, 5 µl of the ligase mixture is mixed with 50 µl of 0.05 m CaCI. then 150 µl of competent cell suspension is added, incubated for 30 minutes at 0 ° C, then 2 minutes at 42 ° C and again 10 minutes at 0 ° C, then 1 A ml of LB-broth is added, incubated for 1 hour 37 ° C and aliquots are plated on LB agar plates containing 100 μg / ml ampicillin. The resulting colonies are transferred to parallel nitrocellulose filters and analyzed for the thymosin gene content by hybridization of the colonies in situ with (P) -labeled oligonucleotides () and (VIII). Colonies that give positive hybridization with both samples are selected and plasmid DNA isolated from them is analyzed using the endonucleases HaeMI and Mspl. Finally, the structure of the recombinant DNTPH1-1-4 is confirmed by determining the part of its nucleotide sequence between the EcoRI and Hindlll sites.

PRI me R 3. Construction of recombinant plasmid DNA pTN12.

DNA MHR m: "d | pC ° G2 (5 μg) hydrolyze with a variety of restriction enzymes (1C1 units each) HY and Hindlll in 100 MULs of buffer R PT .-. 4LN.- 1 5 h of gti Sweat 5 incubator mixture 3 n Spread the walls with a mixture of F ° nol chlorophos - (1: 1} and vectorial Fragment of 3.94 kbp. You are up to 1 g of electrotoem in 1% agarose gel. Then 30 μg of the DNA of the plasmid pTHI1-4 hyd0 Rolled in the same buffer using a mixture of 60 units of each of the restriction enzymes Clal and r-lindlll, and then isolated by electrophoresis in an 8% PAG fragment of 93 i.o., containing the synthetic gene thymosind:.

5 After the end, pygip yoO.15 μg of this Frgzmente with 0.6 μg of Clal / HindIM fragment of pCPG2 pmill in 25 μl of Bufea C in the presence of 50 units. T4 DNA ligase. After 3 hours at 15 ° C, 5 μl of the ligase mixture is used to transform competent E. HB101 cells. The screening of the group is the diblification of chloro-phenyc olustoychiryh. with pj-mpuchmhm sligonuchotndom low frequency presence of synthetic o {rgment, -z

5 timogogin genes. DNA was isolated from hygridizing clones and analyzed using res. ktae Haeil1 and Wspl and the top structure of the /prede /; en | -.-- and nukheptidnoe posleoitioios and mog.ifitsig ::; 0 by the bath method of Maksam-G1.bs of-a.

Etc ; .1 p p 4 KoHCTpVnpOi; .. 0 G Ombi-H1H and / i; -..one. L Ng: g TH Y3.

DN-: g-lhchchidp g (10 gih) sbs- - gy-.z-: t g 70 μl buffer B pЈ: g.trnkt. -.- .-, 5 Clsl and Rgl (20 units) for 1 m Grp. the fragment was led by 2.9 so on. Electrophoiesc of a 1% gel Kaplapko ga rose is measured. 15 μg of plasmid pTHNF314 D DNA is digested with restrictases

0 WatN and Pstl (20 units each) for 1.5 hours at 37 ° C and a fragment of 1.48 kb was isolated containing a promoter of cryptation and mutant HRN of a factor of human tumors. A 0.2 gelled DNA fragment is ligated with an E of 25 µl of buffer C with 0.5 µg of vector DNA. obtained from the plasmid rtH12 with oligonucleotides (IX) and (X) (0.2 μg each) using 50 units. T4 DNA ligae for 15 h at

0-15 ° C. An aliquot of the reaction mixture is used to transform competent E coP cells. Transformants are plated on plates with LB-agar containing ampicillin and n (100 m g / ml) and chloramphenol.

5 (50 vr / ml). The screening of rexmbinanto is performed by hybridization of colonies with (P) labeled ol igonuh-eotide (X). The plasmid DNA of pTH Y314 was isolated from the hybridizing clones, which was analyzed by restriction analysis using the Haelll and Mspl endonucleases and the nucleotide sequence of the junction area of the tumor necrosis factor and thymosin C structural genes.

PRI me R 5. Obtaining a producer strain of a polypeptide with the properties of human thymosin a.

Plasmid pTNU314 transform competent E.coll SG 20050 cells according to the method of Example 2 and obtain a producing strain of a polypeptide with human thymosin a properties.

Example Determination of the productivity of E.coll strain - a producer of a polypeptide with human thymosin a properties.

E.coll SG 20050 / pThy314 cells are grown at 37 ° C in 1 L of LB-broth. containing ampicillin and chloramphenicol (30 µg / ml each) for 20 hours on a rocking chair at a rotation speed of 190 rpm. The cells are centrifuged for 10 minutes at 6000 rpm, then suspended in 15 ml of buffer containing 10% sucrose, 50 mM Tris-HCl. pH 8.0, 0.2 mM NaCl and 0.1 mM phenylmethylsulfonylfluoride. and voiced by pulses for 30 seconds. at a frequency of 22000 Hz for 10 minutes at 4 ° C. The resulting lysate is centrifuged for 15 minutes at 8,000 rpm. The pellet is suspended in 10 ml of 7 M guanidine chloride at 4 ° C for 6 h. After centrifugation, the supernatant is diluted with 10 volumes of cold water, and the precipitated precipitate is separated by centrifugation. The precipitate (according to SDS-electrophoresis in 13% PA-AG of 90% purity chimeric protein — tumor necrosis factor — thymosin 0.7 g wet weight) is suspended in 10 ml of 0.1% BrCN in 80% formic acid. acid, and the mixture is stirred for 16 h at 20 ° C. after which the solvent is removed in vacuo at 30 ° C. The residue is suspended in 2 ml of 7 M urinary, the pH of the solution is adjusted with ethanolamine to 5.0 and shaken with 2 parts at 20 ° C. after which it is diluted with water to a volume of 50 ml and kept under stirring for 12 hours at 4 ° C. The precipitate is removed by centrifugation (30 min at 15,000 rpm). the solution was applied to a column (1.2 x 30 cm) with DEAE DE-52 cellulose equilibrated with 20 mM TEAB (pH 7.9). For elution, a NaCI concentration gradient in the same buffer is used. Desacetyl thymosin "1 is epjurised from the column at a NaCI concentration of 0.2-0.3 M.

Fractions containing peptide. combined, concentrated by lyophilization, and chromatographed in 10 mM ammonium bicarbonate buffer on a column (1x50 cm) with Uluogel AcA 202. Fractions containing thi

five

0

five

0

five

0

five

0

five

moi ai, pooled and lyophilized. 8.5 mg of peptide are obtained, the frequency of which is 95%.

Thus, the proposed method allows to obtain a peptide with human thymosin properties with a biosynthesis level of 8-10 µg / ml of cell culture. At the same time, the technology of its production is greatly simplified by eliminating the stage of induction of protein biosynthesis.

Claims (3)

1. Recombinant pTNU314 plasmid DNA encoding a polypeptide with human thymosin A properties, 3.98 kb in size. and mol.m. 2.57 MD, containing: -Pstl - Sau3AI - DNA fragment of plasmid PTNF314 D, 1.48 kb in size, with a semi-synthetic gene of human tumor necrosis factor and a synthetic translation initiation site: -Clal-Pstl - DNA fragment of plasmid pTH12. size 2.49 TP, with a synthetic gene encoding a polypeptide with the properties of thymosin and. -SauSAI CIal is a synthetic linker. GATCAT1GCCCTGAT TAACGGGACTAGC: - genetic markers: Na - gene / -laktemazy; cat is the chloramphenicol acetyltransferase gene; - tandem promoters of transcription A2 and A3 of the early region of bacteriophage T7, - unique restriction sites, located at a distance to the right of the Kpnl site: BstEl - 405 nucleotides; Bglll - 492 nucleotides; Hindlll - 586 nucleotides. Pst - 3779 nucleotides. 2. Recombinant plasmid DNA pTHI12 - an intermediate product for the construction of recombinant plasmid DNA pTHU314, encoding a polypeptide with the properties of thymosin o. human, size 4.02 kb, containing: - Hindlll - Gal - DNA fragment of plasmid pCPG2, 3.92 Kb in size; - C1A - Hindlll - synthetic gene. coding polypeptide thymosin a person, 0.1 kb in size .: - unique restriction sites located at the following distances to the right of the Bam HI site: Clal - 24 nucleotides: Bgl II - 75 nucleotides; Hindlll - 118 nucleotides: Ncol - 729 nucleotides; Pstl - 2573 nucleotides; - tandem transcription promoters A2 and A3 of the early region of bacteriophage T7, synthetic translational initiation site, phage A transcription terminator; 131707078 14 - genetic markers: 3. Strain of Bscterium Fscherlchla coii LA - D-lactamase gene: VKPM B-5056 - producer of the polypeptide with cat - the gene for chloramphenicol acetyptrans - with thymosin properties of chepovekl. Ferae.