A method of obtaining a transgenic animal expressing breast granulocyte colony-stimulating factor human and hybrid gene h-gm-1 for implementing the method

 

(57) Abstract:

Human granulocyte colony-stimulating factor (G-CSF) is produced by expression of recombinant proteins in mammary gland of transgenic animals. To obtain protein G-CSF human use milk of transgenic animals. Created original gene construct G-CSF person on the basis of available genomic copies of DNA and regulatory sites of genes milk proteins that enable the efficient secretion of G-CSF with milk of transgenic animals. The invention can be used in immunology. 2 c.p. f-crystals, 3 ill.

The invention relates to biotechnology.

A known method of receiving granulocyte colony-stimulating factor (G-CSF) from sources such as blood, where it is the norm there. This method is time-consuming and roads due to the low concentration of biologically active substances and a limited number of raw materials (Werner RG, Berthold W; Purification of proteins produced by biotechnological process. Arzneimittelforschung 1988 Mar; 38(3):422-8). Another danger of this method is the possibility of the presence in the final product infectious for human agents.

Another source of useful biologically active substances are poluchenii method is associated with certain problems. Synthesis system prokaryotes cannot properly carry out post-translational processing of proteins - folding, protein modifications, such as glycosylation, siliconizing, and so on, it is Shown that for the functional activity of G-CSF is important glycosylation. Glycosylated form of G-CSF in 2 times exceeds the ability to stimulate the growth of colonies of granulocytes and 20 times faster leads to a positive effect compared to the existing commercial deglycosylated recombinant drug Filgrastim (Nissen S. Glycosilation of recombinant human granulocyte colony-stimulating factor for stability and potency. Eur. J. Cancer, 1994, 30A, Suppl 3: S12-4). Using some processes, it is possible to separate substances to increase the yield of biologically active protein, such as insulin, the PUK. However, for most substances, this method is not applicable. The best results for different time of the protein in the culture of mammalian cells. All necessary modifications normally occur, but the concentration of the desired protein is low, and the process of cultivation expensive and requires a high technological support.

A known method of obtaining biologically active substances using transgenic mammalian animals, such as rabbits, sheep, Krsti derived substances native. A limitation of the method is the high cost of raw materials and low concentration in the produced substances, as well as the adverse effects produced protein in the transgenic organism of the animal (I. L. Goldman et al. Transgenic farm animals: the expression of foreign genes. Biotechnology, 1996, No. 9, S. 3-23).

A known method for producing transgenic animals expressing alpha-1-antitripsin, factor IX (E. D. Clark, R. Leis, PPL Therapeutics Ltd., "A method of obtaining a transgenic sheep", patent of the Russian Federation Ru #2085587 C1, 1986) and gamma-interferon person (Lagutin, O. C. with al, "a Hybrid gene BLG-HIFN-G for the expression of gamma-interferon person in the mammary gland of transgenic animal", patent of Russian Federation N 2084525 C1, 1997) in the mammary gland of the lactating females.

The authors of the present invention, a method for producing transgenic animals, providing for the design of genetic constructs plasmid DNA that encodes a fusion protein of hG-CSF under the control of promoters of genes of milk proteins that provides secretion of the recombinant protein in the mammary gland of the lactating females, microinjection fragment into the pronucleus of a fertilized egg and transplantation of ova in PMU, as well as a high concentration of a substance in raw materials.

Human granulocyte colony-stimulating factor (G-CSF) stimulates the formation of neutrophils and is widely used in combination with chemotherapy in the treatment of cancer patients, patients neutropenia and anemia.

Currently abroad recombinant G-CSF obtained from bacterial cells in the form of Neupogen and Filgrastim. The use of prokaryotes for the preparation of recombinant proteins, as it is known, provides a number of post-translational modifications, in particular glycosylation, protein folding and removal of excess methionine from the N-Terminus of the protein, resulting in reduced biological activity of the final product. Moreover, its cost is very high. Recombinant proteins derived from transgenic eukaryotic organisms, for example, by targeting expression in the milk of transgenic animals, in particular rabbits, not have these disadvantages. The use of transgenic animals allows with minimal cost to obtain a sufficient amount of the drug in the shortest possible time. The concentration of the desired protein in milk can reach 5-15 g/l, which amounts to 50% of the total Belk is the growing genomic copies of DNA providing effective secretion of G-CSF with milk of transgenic animals. Transgenic rabbits on the basis of methods developed in the Scientific and industrial biotechnology the center for animal breeding of the RAAS.

Created by the authors of the hybrid gene for the expression of G-CSF in the milk of transgenic animals are characterized by the following features:

- ensure the expression of human protein G-CSF in the mammary gland of transgenic animals;

- consist of:

- Kpn fragment 1 and Cla 1 size 0,9 thousand p. N. 5'-flanking region of the gene beta-casein bull containing the promoter of the gene BLG (beta-lactoglobulin bull);

fragment size 1.5 thousand p. N., containing a genomic copy of the gene G-CSF person;

fragment size of 0.35 thousand p. N., containing the 3'-flanking region of the gene beta-casein bull;

- plot of polylinker containing a unique recognition sites of the restriction endonucleases EcoR I, Hind III, Sal I, BamH I, Xba I, Not 1. Diagram of the vector hybrid gene hGM-l shown in Fig .1. The sequence of the hybrid gene is shown in Fig. 3.

The creation of a hybrid gene G-CSF person and obtaining transgenic animals is illustrated by the following examples:

EXAMPLE 1.

Creating gibridnogo the gene of beta-lactoglobulin bull with flanking 5' and 3' sequences, unique sites restricted Kpn 1 and Cla 1 isolated fragment of the 5'-flanking region containing the promoter of the gene BLG and its signal peptide. From this vector by polymerase chain reaction with specific primers was isolated 3'-flanking gene BLG.

5'-fragment was periglomerular in the site restrictase Cla 1 in the vector phGCSF2 containing a genomic copy of the gene G-CSF person, before the ATG site of the gene G-CSF. Correct cloning and preservation of the reading frame was verified by the method of sequencing of fragment design, amplified by specific primers CCT GCA GAG CTC AGA AGC GTG and AGG CGG CTC TCC CAT CCT GGG. The resulting structure called p5LG-GCSF.

the 3'fragment was periglomerular in the site restrictase Xho 1 in the vector p5LG-GCSF, after the stop codon coding portion of the gene G-CSF. The resulting vector was checked for the correctness of the coding sequence by the method of sequencing key areas of the hybrid gene. The resulting vector was named phGM-1.

In the preparation of the fragment for microinjection his cut of 20 μg vector DNA phGM-1 endonucleases PKK 1 and Not 1. Then the fragment was isolated from the mixture by the method of fractionation on an agarose gel and was purified by phenol-chloroform method. Final,1 mm EDTA, pH 8.0).

EXAMPLE 2.

Obtaining transgenic rabbits

As donors zygotes use of Mature female rabbits of the chinchilla breed at the age of 5-7 months. For inducing superovulation of donors used gonadotropin serum of pregnant mares (Sargon, Czech Republic). Each donor is injected ME 100 of Sargon, 48-72 hours female pair with the male and induce ovulation intravenous 200 ME chorionic gonadotropin (Moscow endocrine plant). To retrieve zygotes females donor operational use washing oviduct in vivo (Adams C. E. Egg transfer in the rabbit. The Mammalian egg transfer. Weight Exploration: CRC Press, 1982. P. 29-48). As use anesthetics ketamine 5%, rompun 2%. Anesthetized animal is fixed on the operating table and vypivaut coat in the area of the belly. Reproductive tract naked through the incision along the white line of the abdomen. In the funnel of the oviduct injected polyethylene catheter in the wall of the uterine horn with a metal cannula with stylet make the hole, then into the lumen of the uterus impose warm manipulation environment and direct it to the current through the connection of the uterus with fallopian tubes in the oviduct.

Search fertilized oocytes (zygotes) was performed on binocular loupe Nikon when growing the mustache (Brem-G. et al. Production of transgenic mice, rabbits and pigs by microinjection into pronuclei// Zuchthyg, 1985, v. 20, p. 251-252; R. E. Hammer et al. Production of transgenic rabbits, sheep and pigs by microinjection. Nature. 1985, V. 315, P. 680-683). Microinjection is carried out in a special chamber filled with culture medium (PBS with 5% fetal serum). The camera consists of two parallel siliconized cover slides between which the droplet manipulation of the environment in the form of a column. All the rest of the space filled with mineral oil (Sigma, d=0.84 g/ml). Intended for injection of the zygote is placed in a column manipulation of the environment, keep the glass pipette and appreciate the pronuclei using an inverted microscope Axiovert 35 at 400x magnification. The DNA solution is injected using microinjection needle extended from the capillary tubes with microfilaments (thin-walled refractory glass, diameter 1 mm) on a vertical puller (NGOs "Biofiber RAS).

Microinjection needle was filled with DNA solution using capillary forces, and then installed in the optical drive. The position of both micro tools (fixing pipette and microinjection needle) regulate the use of micromanipulators (NGOs "Biofiber RAS). Microinjections tried to increase pronucleuses. Injected zygote is incubated for 30-60 min to remove damaged. It degenerated attributed zygote with fragmented cytoplasm and with damaged cytoplasmic membrane. The zygote, with normal appearance of the transplant recipient the DOE-rabbit, whose reproductive cycle is synchronized with the cycle of the donor. Female recipients were paired with watertolerant male and injected human chorionic gonadotropin at the same time, when paired donor female with a full male. Possible preparation of the recipient immediately prior to embryo transfer. Embryos transplanted into the oviduct of female recipients surgical way. For transplanting the zygote use sterile catheters for transplant embryos (Biomedical, Russia). The recipient through the funnel of the oviduct is injected with a catheter containing a manipulation environment with zygotes. Each recipient was transplanted 15-20 zygotes, distributing them equally between the fallopian tubes.

After embryo transfer, each DOE-rabbit-recipient is in a separate cage for the duration of pregnancy. Within 7-10 days is carried out rehabilitation of the surgical suture. On 14-16 days after embryo transfer by palpation establish if the button. The kindling waiting for 29-30 days after embryo transfer. In case of delay of kindling on the next day after the expected date for the purpose of facilitating delivery of intravenously injected 3 IU of oxytocin. Were accurate account of the number of newborn rabbits. After 5-7 days of all rabbits were labeled and simultaneously took the ear tissue samples for DNA analysis on the integration of the transgene.

The presence of integration was determined by polymerase chain reaction (PCR) with specific primers GCA CAG CCT GTA GGT GGC ACA and CCT GCA GAG CTC AGA AGC GTG. Positive PCR samples were confirmed by the method of blot-hybridization (Fig. 2).

As a result of the research was received 2 living transgenic rabbit (N 7 and N 25), and 3 stillborn.

The list of references.

I. L. Goldman et al. Transgenic farm animals: the expression of foreign genes. Biotechnology, 1996, No. 9, S. 3-23

Hammer R. E. et al. Production of transgenic rabbits, sheep and pigs by microinjection. Nature. 1985, V. 315, P. 680-683.

E. D. Clarke, P. of Las, PPL Therapeutics Ltd., "A method of obtaining a transgenic sheep", patent of the Russian Federation Ru #2085587 C1, 1986.

Nissen S. Glycosilation of recombinant human granulocyte colony-stimulating factor for stability and potency. Eur. J. Cancer, 1994, 30A, Suppl 3: S12-4.

Adams C. E. Egg transfer in the rabbit. The Mammalian egg transfer. Weight Exploration: CRC Pr Hosp Pharm 1989 Sep; 46(9):1834-44.

Brem-G. et al. Production of transgenic mice, rabbits and pigs by microinjection into pronuclei// Zuchthyg, 1985, v.20, p. 251-252.

1. Hybrid gene h-GM-1 for the expression of granulocyte colony-stimulating factor human (G-CSF) consisting of a fragment of 0.9 thousand p. N. 5'-flanking region of the gene of betacatenin bull containing the promoter of the gene of beta-lactoglobulin bull (BLG); fragment size 1.5 thousand p. N., containing a genomic copy of the gene granulocyte colony-stimulating factor (G-CSF) human; the fragment size of 0.35 thousand p. N., containing the 3'-flanking region of the gene beta-casein bull; and shown in Fig.3, providing for efficient secretion of granulocyte colony-stimulating factor human (G-CSF) in the mammary gland of transgenic animal.

2. A method of obtaining a transgenic animal expressing breast granulocyte colony-stimulating factor human, by microinjection hybrid gene h-GM-1, described in paragraph 1, into the pronucleus of a fertilized egg and the subsequent transfer of the egg in a sexual way, pseudoelement females.

 

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