RU2385938C1 - AGPopt GENE OF ANGIOPOIETIN-1 - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: invention relates to the preparation of genetically engineered construct for transgene expression in mammal cells in vitro and in vivo. The modified sequence of gene of angiopoietin-1 (Angl) is offered. The sequence is designed for transfection of mammal cells for expression of angiopoietin-1 therein. Production of angiopoietin-1 in case of using the optimised gene AGPopt is 1.6 times more than when using the natural gene hAGP. Herewith, the content of angiopoietin-1 in a culture medium of transfected cells makes not less than 0.5 mcg/ml. ^ EFFECT: invention can be used in medicine. ^ 3 dwg, 1 tbl, 1 ex

Description

The invention relates to the field of biotechnology, and in particular to a technology for producing genetic engineering constructs for the expression of transgenes in mammalian cells in vitro and in vivo for the purpose of their use in cell engineering and gene therapy, namely, a fragment of a vector — a modified sequence of the angiopoietin-1 gene (Ang1), and is intended for transfection of mammalian cells with the aim of expressing angiopoietin-1 in them.

Angiopoietin-1 is a vessel stabilization factor that regulates the formation of a full-fledged mature vessel by attracting smooth muscle cells and reducing vascular permeability (Brindle NP, Saharinen P., Alitalo K. Circ. Res., 2006, Vol.98, p. 1014-1023) . Angiopoietin-1 also inhibits EC proliferation and helps to attract pericytes. For the production of Angl, DNA containing the natural human angiopoietin-1 gene is used (Siddiqui AJ, Blomberg P, Wärdell E, Hellgren I, Eskandarpour M, Islam KB, Sylvén C. Biochem Biophys Res Commun. 2003, Oct 24; 310 (3): 1002-9).

The technical problem solved by the authors of the present invention was the modification of the sequence of the angiopoietin-1 gene so that when it is included in the eukaryotic vector, a higher level of expression of the named protein is provided in cells transfected with the named vector.

The technical result was achieved by modifying the natural sequence of the angiopoietin-1 gene, which was based on the degeneracy of the genetic code. The sequence was modified so that the triplets of nucleotides encoding some of the amino acids of the angiopoietin-1 protein sequence were found most frequently in human genes. We used data published in open sources on the frequency of occurrence of codons (http://del.mediaglyphs.org/mg/bf/nucs_aa.html).

Figure 1 shows the nucleotide sequence of the AGPopt gene (top lines) in comparison with the nucleotide sequence of the natural human angiopoietin-1 gene (middle lines). The amino acid sequence of the encoded protein product is also shown (bottom lines). The region encoding the protein is highlighted in bold. The triplets of nucleotides that have undergone optimization are emphasized. The signals of ATTTA mRNA destruction and AATAAA polyadenylation are darkened.

Figure 2 shows the functional map of the plasmid pC4W-AGPopt.

Figure 3 shows the Western blotting of the proteins of the culture medium of HEK293 cells transfected with plasmid pC4W-hAGP (lane 2) and plasmid pC4W-AGPopt (lane 3). Lane 1 contains 20 ng of recombinant human angiopoietin-1.

The synthesis of the gene and plasmid was carried out using standard technology and equipment used to solve similar problems in genetic engineering (Watson, JD, Gilman, M., Witkowski, J., Zoller, M. - Recombinant DNA, Scientific American Books, New York, 1992).

An optimized nucleotide sequence encoding human angiopoietin-1 was designed as follows. Using the codon frequencies found on the website http://del.mediaglyphs.org/mg/bf/nucs_aa.html, among triplets of nucleotides encoding the amino acids of the protein sequence of the natural angiopoietin-1 gene, those that are found in human genes were detected most rarely. Identified rare triplets, as well as neighboring triplets, were exchanged for triplets encoding the same amino acid, but which are most often found in human genes. In addition, in order to increase the stability of angiopoietin-1 messenger RNA by the same methods, the ATTTA mRNA destruction signals and AATAAA polyadenylation signals (Barreau C., Paillard L., Osborne BH Nucleic Acids were removed from the protein coding region of the natural angiopoietin-1 gene Research, 2005, Vol. 33, p. 7138-7150). As a result, due to the degeneracy of the genetic code, all amino acids of the natural angiopoietin-1 gene remained unchanged. The resulting nucleotide sequence is presented in figure 1.

Angiopoietin-1 cDNA was obtained by RT-PCR using total RNA from the human liver as a matrix and cloned into pcDNA3 expression vector (Invitrogen, USA) to obtain pcDNA3-hAGP plasmid and into pC4W expression vector (MONA LLC, Russian Federation) to obtain the plasmid pC4W-hAGP. Plasmid pC4W-AGPopt carrying the optimized AGPopt gene was obtained by genetic engineering, as described below.

In the first stage

- chemically synthesized pair of oligonucleotides

VEC01 (AGCTTGGCATTCCGGTACTGTTGGTAAAGCCACCATG) and

VEC02 (GTACCATGGTGGCTTTACCAACAGTACCGGAATGCCA) was annealed, mixed and cloned into the restriction enzyme digested HindIII and Ass65I vector pUC19 to obtain the intermediate plasmid pUC-KNA;

- chemically synthesized pair of oligonucleotides

VEC05 (AGCTTGGCATTCCGGTACTGTTGGTAAAGCCACCATGG) and

VEC06 (GATCCATGGTGGCTTTACCAACAGTACCGGAATGCCA) was annealed, mixed and cloned into the HindIII and BamHI restriction enzyme digested pBluescriptII-SK ⁺ vector to obtain the intermediate plasmid pBSK-KNB.

In the second stage, a series of amplification reactions was carried out using the pcDNA3-hAGP plasmid as a matrix and pairs of chemically synthesized oligonucleotides

As a result, DNA fragments were obtained, respectively, AGP1-2, AGP39-40, AGP41-42, AGP43-44, AGP45-46, AGP47-48, which were purified by agarose gel electrophoresis and electroelution.

In a third step, an amplification reaction was carried out using AGP39-40 and AGP41-42 DNA fragments as a template and AGP39F and AGP42R oligonucleotides as primers; as well as an amplification reaction using AGP45-46 and AGP47-48 DNA fragments as a template and AGP45F and AGP48R oligonucleotides as primers. As a result, DNA fragments, respectively, AGP39-42 and AGP45-48, were obtained, which were purified by agarose gel electrophoresis and electroelution.

In a fourth step, an amplification reaction was carried out using DNA fragments AGP39-42 and AGP43-44 as a template and oligonucleotides AGP39F and AGP44R as primers; and also an amplification reaction using AGP43-44 and AGP45-48 DNA fragments as a template and AGP43F and AGP48R oligonucleotides as primers. As a result, DNA fragments, respectively, AGP39-44 and AGP43-48, were obtained, which were purified by agarose gel electrophoresis and electroelution.

In a fifth step, an amplification reaction was carried out using AGP39-44 and AGP43-48 DNA fragments as a template and AGP39F and AGP48R oligonucleotides as primers. As a result, an AGP39-48 DNA fragment was obtained, which was purified by agarose gel electrophoresis and electroelution.

In the sixth step, the AGP1-2 DNA fragment was digested with restriction enzymes BamHI and EcoRI and cloned into the pBluescriptII-SK ⁺ vector digested with the same restriction enzymes to obtain the intermediate plasmid pBSK-AGP1-2;

chemically synthesized pairs of oligonucleotides:

a fragment of the plasmid pcDNA3-hAGP was cloned into the pBSK-KNB plasmid digested with restriction enzymes NcoI and BamHI to obtain the intermediate plasmid pBSK-AGPN-8. The AGP39-48 DNA fragment was digested with restriction enzymes BamHI and SmaI and cloned into the pBluescriptII-SK ⁺ vector digested with the same restriction enzymes to obtain the intermediate plasmid pBSK-AGP39-48. The insertion of the corresponding DNA sequences was confirmed by sequencing.

At the seventh stage

cloned into the plasmid pBSK-AGP31-34 digested with restriction enzymes PstI and XbaI to obtain the intermediate plasmid pBSK-AGP31-38. The HindIII - NcoI fragment of the plasmid pBSK-AGP39-48 was cloned into the plasmid pBSK-AGPN-8 digested with restriction enzymes HindIII and NcoI to obtain the intermediate plasmid pBSK-AGP39-8. The insertion of the corresponding DNA sequences was confirmed by sequencing.

In the eighth stage

- HindIII - EcoRI fragment of plasmid pUC-KAGP3-2 and EcoRI - NcoI fragment of plasmid pcDNA3-hAGP were cloned into plasmid pBSK-AGPN-8, digested with restriction enzymes HindIII and NcoI, to obtain the intermediate plasmid pBSK-KAGP;

- chemically synthesized pair of oligonucleotides

AGP17S (GGACACCCTGAAGGAGGAGAAGGAGAACCTGCA) and

AGP18A (GGTTCTCCTTCTCCTCCTTCAGGGTGTCCAGCT) and the SbfI - HindIII fragment of plasmid pUC-AGP19-30 were cloned into plasmid pUC-AGP9-16, digested with restriction enzymes SacI and HindIII, to obtain intermediate plasmid pUC-AGP9-30;

- Acc65I — The Eco47III fragment of plasmid pBSK-AGP31-38 was cloned into plasmid pBSK-AGP39-8 digested with restriction enzymes Acc65I and SmaI to obtain the intermediate plasmid pBSK-AGP31-8.

The insertion of the corresponding DNA sequences was confirmed by restriction analysis and sequencing of the obtained plasmids.

In the ninth step, EcoRI — the BsrGI fragment of the plasmid pUC-AGP9-30 and the Acc65I-BamHI fragment of the plasmid pBSK-AGP31-8 were cloned into the plasmid pBSK-KAGP digested with restriction enzymes EcoRI and BamHI to obtain the intermediate plasmid pBSK-KAGPopt. The insertion of the corresponding DNA sequences was confirmed by restriction analysis of the obtained plasmid.

In the tenth step of HindIII-BamHI, a fragment of the pBSK-KAGPopt plasmid was cloned into the highly efficient pC4W expression vector digested with the restriction enzymes HindIII and BamHI to obtain the plasmid pC4W-AGPopt. The correct insertion of the optimized AGPopt gene and the correspondence to the design of its nucleotide sequence were confirmed by restriction analysis and sequencing of the obtained plasmid. A functional map of the plasmid pC4W-AGPopt, designed for expression of the human angiopoietin-1 gene in mammalian cells, is presented in Fig.2.

The effectiveness of the claimed invention is illustrated by the following example.

Example. HEK293 cells seeded in the wells of a 24-well plate (50,000 cells per well) in DMEM containing 2% fetal calf serum were transfected with the pC4W-AGPopt plasmid carrying the optimized human angiopoietin-1 gene (AGPopt), or the control plasmid pC4W-hAG carrying the natural human angiopoietin-1 gene (hAGP). Transfection was performed using 1 μg DNA and 1 μl Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer's protocol. 24 hours after transfection, the culture medium was replaced with fresh. The accumulation of angiopoietin-1 in the culture medium over the next 48 hours was analyzed.

Western blotting was used to observe the expression of angiopoietin-1 after cell transfection. Protein electrophoresis in a 13% polyacrylamide gel was carried out under denaturing conditions in the presence of sodium dodecyl sulfate without the addition of beta-mercaptoethanol. 30 μl of culture medium was applied to the gel tracks.

The transfer of proteins from the gel to the PVDF membrane (Millipore) was carried out under semi-dry conditions in a standard buffer. After transfer, the membrane was incubated in a 5% skim milk solution in TBS buffer (Tris buffer saline) with 0.001% Tween 20 (Pierce) overnight at 4 ° C. Then, incubation was performed with murine monoclonal antibodies against human angiopoietin-1 from R&D Systems (USA), which were diluted 500 times in 5% skim milk in TBS buffer with 0.001% Tween 20. The membrane was washed three times in 5% - skim milk solution in TBS buffer with 0.001% Tween 20 for 15 minutes and incubated with peroxidase-labeled rat antibodies against mouse immunoglobulins in 5% skim milk with 0.001% Tween 20 at room temperature for 60 minutes . After that, the membrane was treated with a reagent kit of a two-component ECL system (Pierce), exposed with a Bio Max X-ray film (Kodak), and densitometry was performed. The results are presented in the table and figure 3, which shows Western blotting of the proteins of the culture medium of HEK293 cells transfected with plasmid pC4W-hAGP (lane 2) and plasmid pC4W-AGPopt (lane 3). (Lane 1 contains 20 ng of recombinant human angiopoietin-1.)

Densitometric analysis of Western blotting of proteins of the culture medium of transfected cells Plasmid Integrated Protein Spot Signal pC4W-hAGP 45059 pC4W-AGPopt 74312

Semiquantitative densitometric analysis showed (table) that the production of angiopoietin-1 in the case of using the optimized AGPopt gene is 1.6 times greater than in the case of using the natural hAGP gene. Comparison of the signal from cells transfected with plasmid pC4W-AGPopt (Fig. 3, lane 3), on the one hand, and the signal from recombinant human angiopoietin-1 (lane 1), on the other hand, allows us to conclude that the content of angiopoietin-1 in the culture medium transfected cells is at least 0.5 μg / ml.

Claims (1)

Gene angiopoietin-1, having the nucleotide sequence shown in Fig.1.

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