Fragment to dna encoding glycoprotein tcf-ii

 

(57) Abstract:

The invention relates to biotechnology. Obtaining the nucleotide sequence of the fragment to DNA encoding a new glycoprotein F-II of human origin, with antitumor activity, activity of inducing the differentiation of leukemia cells, the activity increased cellular immunology, activity to stimulate the growth of endothelial cells of blood vessels, as well as activity to stimulate the growth of genocidal. 1 C.p. f-crystals, 3 ill.

The invention relates to a DNA sequence that encodes a glycoprotein of human origin.

Glycoprotein exhibits cytotoxic activity against different lines of tumor cells, but not against normal cells, acts as a new antitumor cytotoxic factor, factor contributing to the differentiation of the leukaemic cells, a factor that boosts cellular immunology, factor contributing to the growth of endothelial cells of blood vessels, factor contributing to the growth of hepatitas.

In patent application laid Japan N 64-10998 described the entire amino acid sequence and the nucleotide sequence in the set is tov human origin and characteristics: molecular weight 360001000, isoelectric point over 10.5.

According to the invention Primera sequence of TCF-II was deduced from its cDNA, which was cloned sort cDNA library, obtained using mRNA isolated in pure form from the total number of total RNA, which in turn was extracted from cells IMR-90 fibroblasts embryo of man according to the following method.

(1) Extraction of poly(A) RNA from cells UMR-90.

All RNA was prepared by a method involving the use of guanidine thiocyanate and cesium chloride. Of cells, IMR-90, taken in the amount of 200 million, which is cultivated in the environment of the proposed Eagle (Eagle) and modified Dulbecco (Dulbecco) containing 5% newborn calf serum (NBCS). Cells IMR-90, subjected to averaging, represent a suspension in 28 ml of 6M guanidine thiocyanate, also containing sodium citrate (5 mm), 0.5% drug Sarkosyl and beta mercaptoethanol (0.1 M). In centrifuge tubes, made of polyallomer, placed in 4 ml of 5.7 M solution of cesium chloride containing 0.1 M ethylenediaminetetraacetic acid. Then to a solution of cesium chloride was added 7 ml of homogenized cell suspension and implement centrifugable centrifugation of the particles are washed twice with 95% ethanol and dissolved in 200 µl of buffer solution Tris HCl (10 mm, pH 75,5) containing 1 mm ethylenediaminetetraacetic acid, by heating at 65oC for 5 minutes the resulting solution is then marked as the solution of total RNA. Poly (A) RNA isolated in pure form from the total (all) RNA by chromatography in a column of oligo (dT) cellulose. The solution of total RNA is poured into a column of oligo (dT) cellulose, are in equilibrium with 10 mm buffer solution Tris HCl (10 mm, pH 7,4) containing 1 mm ethylenediaminetetraacetic acid and 0.05% SDS. The resulting solution are outlined below as a solution of poly(A) RNA.

(2) Synthesis of cDNA library.

Double helix synthesize cDNA using poly A RNA from paragraph (1) as a template, and using proprietary tools for the synthesis of cDNA (produced by Pharmacia Co. Ltd), and additionally attach the adaptor EcoR 1. The method of synthesis carried out according to the recommendations specified in the company except for the addition (at the stage of synthesis of single DNA helix) reverse transcriptase inhibitor (40 units in the reaction mixture, produced by Life Science Co. Ltd), obtained from the culture of non-spherical pathogenic virus that kills bone marrow birds.

(3) preparation of a cDNA library.

Complem the sector of gt 10 phage. This cDNA in the amount of 3.3 µg synthesized from poly A RNA, dissolved in 150 μl of buffer solution 66 mm Tris-HCl (pH 7,6) for the chromatographic column containing spermidine (1 mm), magnesium chloride (10 mm), dithiothreitol (15 mm), and bovine serum albumin (0.2 mg/ml). This solution is in the amount of 6.2 μl is mixed with 1 μg EcoR 1 part of the phage (vector gt 10), followed by precipitation with ethyl alcohol. Recombinant phage DNA, including both components, gt 10 and cDNA prepared as follows. The above precipitate was transferred into a slurry using 9 CML buffer solution, designed for the chromatographic column, and left for incubation over night at 16oC with the addition of 1 ál of 10 mm of adenosine triphosphate, and 1 μl of T4 ligase DNA in the amount of 350 units per 1 ml.

(4) Screening the library DNA.

(i) Preparation of oligonucleotide samples.

For preparation of samples comprise a mixture of (labeled 5' terminus) complementary oligonucleotide with the characteristics of the 17 Mer (mer) (a combination of 384 species) of the corresponding amino acid sequence from Va11to Pro6in the N-terminal amino acid sequence of the beta chain of factor TCF-II, with a polynucleotide ke shown as follows: additional spiral, used as the standard (a mixture of 384 species):

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(ii) Screening recombinant phage.

500 thousand disks phage receive laboratory packaging solution of recombinant phage DNA prepared under paragraph (3), using the drug Gigapack Gold (Stratagene) and then infect E. coli E. coli 600hf1. After the adsorption is carried drives phage on the filter brand Hybond-N, Amersham) these drives are denatured with alkali, neutralized and subjected to heat treatment at 80oC for 2 h Hybridization according to the method, which was developed by bell and al., performed using the mixed samples were obtained under paragraph (1). One of the clones, which was supposed to contain a fragment of TCF-II was found among the positive clones detected by the first sort.

(5) Cloning of full length cDNA factor TCF-II.

Internal amino acid sequence (single-letter code): () NYMGNLSQTRSGL and () TSXSVYGWGYTGLINYDGLL (amino acid X is not identified) is determined from the alpha and beta chains of TCF-II by treatment with leilajapanese provided subsequent mapping) fragments. N-terminal amino acid sequence of a beta chain of TCF-II coincided with analogichnye internal consistency and factor TCF-II had, respectively, and alpha - and beta-chains hHGF. Therefore, it was concluded that the factor TCF-II is a consequence of the expression of one gene family hHGF. Were reported data on cDNA libraries of genes hHGF from the placenta and liver, respectively (3-u). Comparison of full primernih sequences derived from both cDNA, revealed differences of amino acids by 14 positions in their sequence. Based on these data, the assumption has been made about the presence of the gene family hHGF. Region, identical to cDNA (DNA) hHGF gene as from the placenta, and liver, were selected as primernih (primer) sequences for the polymerase chain reaction (Polymerase Chain Reaction-PCR). Chemically synthesize identical oligonucleotides both cDNA genes hHGF at non-coding positions 5' and 3'. Sorting cDNA factor TCF-II conducted by PCR using these oligonucleotides as primers (primer). Primer Sa1-77, with the site of cleavage limiting enzyme Sa1 I, and primer Sph 2203 with the plot of the cleavage limiting enzyme Sph1, synthesized using a reagent that promotes DNA synthesis (produced by Applied Co., Ltd.). Below these primers are shown as follows:

primer Sa1 - 77: 5'-GGTCGACTAGGGACTGACTCCGAACAGGATTC-3' Sa1 I

primer Sph 2203: 5'-GGCATGCACAGTTG (PCR), ál:

Complementary DNA (cDNA) synthesized as described in paragraph (2) and dissolved in 150 μl of buffer solution a chromatographic column - 1

20 μm primer Sa1-77 - 2,5

20 μm primer Sph 2203 - 2,5

10 portions of the reaction solution to the reaction of the PCR buffer solution on the basis of 100 mm Tris HCl with a pH of 8.3, containing 500 mm KCl, 15 mm MgCl, and 0.1% (wt.h./about.h) gelatin) - 10

A mixture of 1.25 mm dGTP, dATP, dTTP, dCTP - 16

Amp1i Tag (5 per 1 μl, produced by TAKARA SHUZO) AND 0.5

Distilled water is 67.5

Then the above solutions are mixed in vitro microfuge volume of 0.5 ml, and the liquid surface cover mineral oil in the amount of 100 µl (produced by Sigma Co., Ltd). The PCR reaction is performed using the system Quick Thermo (produced by Japan Genetics Co., Ltd). After pretreatment at 94oC for 7 min 35 repeat three-stage reaction, including: 1) heat treatment at 55oC for 3 min; 2) reaction involving the polymerase at 72oC for 2 min, and 3) reaction denaturirovannyj at 94oC for 2 minutes Then the reaction mixture is heated at 55oC for 3 min, and then 72oC for 11 min, then cooled it to room temperatures by electrophoresis using agarose gel get a DNA fragment, consisting of approximately 2.3 Kb bases (Kirobases) and recognized as the desired cDNA factor TCF-II. Next, the DNA obtained from the four tubes containing the above-mentioned reaction mixture, precipitated with ethyl alcohol and hydrolyzing enzymes Sa1 I and Sph I. the result of gel electrophoresis agarose using DE81 filter paper (produced by Watmen Co., Ltd) was obtained DNA fragment with approximately 2.3 Kb.

(ii) Subclavian.

The DNA fragment of 2.3 Kb) containing Sa1 I and Sph I sites, obtained according to paragraph (1), built using (ligation) buffer for ligation (produced by TKARA SHUZO) in the plasmid vector pUC18 (produced by Japan gene Co., Ltd), treated with enzymes Sa1 I and Sph I. Recombinant DNA transferout Esherichia coli Co1i DH5 on the method of firm BRL Co., Ltd. Managed to get more than 20 subclones.

(iii) determining the base sequence.

The sequence of bases in the resulting subclones determine dideoxy method using sequenase (version 2.0 manufactured by TOYOBO). Incorrectly incorporated nucleotides on the drug Ampi Tag (produced by TAKARA SHUZO) were corrected by analyzing the nucleotide sequence of the bases in , is received on the above-mentioned method, and the amino acid sequence deduced from this nucleotide sequence.

The nucleotide sequence contains 2172 base pairs (p. O.) from the PBX starting codon to codon TAS termination of transcription. After amino acid translation factor TCF-II contains 723 amino acids. It has been assumed that the amino acid sequence from the first methioninamide (Met) to 29th alanine (A1a) residues is a signal sequence. As shown in Fig.1, the factor TCF-II is a polypeptide containing the circuit and connected by a disulfide bond, which is first synthesized as a single chain. Since N is the closure of the chain of factor TCF-II was blocked, it could not be identified. However, the N-end amino acid sequence of the chain , as well as several internal amino acid sequence of factor TCF-II were determined as mentioned above.

The obtained nucleotide cDNA sequence of TCF-II is very similar to the corresponding sequence in hHGF, which was established Miyazawa al. However, in cDNA factor TCF-II lacking the codons five amino acid residues (F-L-P-SS) from Phe162to Ser<'s the point about that cDNA factor TCF-II is one of the new varieties of hHGF gene family.

In Fig. 3 presents a comparison of the amino acid sequence TCF-II, derived from the above-mentioned nucleotide sequence and amino acid sequence corresponding to the gene HGF (according to Miyazawa al. ).

In Fig. 1 and 2 respectively shows the nucleotide sequence to the DNA and amino acid sequence of factor TCF-II, derived from the specified base sequence.

In Fig.3 provides a comparison of the amino acid sequence TCF-II, derived from the above-mentioned nucleotide sequence and amino acid sequence of the gene hHGF reported by Miyazawa and researcher.

Obtained according to the invention DNA can be used to obtain a glycoprotein with antitumor cytotoxic activity, inducing differencei cell line leukemia activity, enhancing cellular immunology activity that promotes cell growth in vascular endothelium activity.

1. The cDNA fragment encoding the glycoprotein TCF-II and having the nucleotide sequence I presented in C.

2. The cDNA fragment is

 

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