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Recombinant goat fetal fibroblast for expressing antithrombin III

A technology of fibroblasts and antithrombin, applied to cells modified by introducing foreign genetic material, methods based on microorganisms, microorganisms, etc., can solve the problem of loss of foreign genes, difficulty in maintaining stable inheritance of foreign genes, and unfavorable good Problems such as high-efficiency expression of the target protein in transgenic shape, to achieve the effect of avoiding uncertainty and high expression efficiency

Inactive Publication Date: 2013-01-30
QINGDAO SAMUELS INDAL & COMML
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Problems solved by technology

However, due to random integration and other factors, the transgenic cell lines (embryos) produced by GTC are difficult to maintain the stable inheritance of exogenous genes. Generally, exogenous genes will gradually be lost with the passage of cell lines or the breeding of transgenic animals, which is not conducive to good transgenic Shape maintenance and high-efficiency expression of target protein

Method used

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Embodiment Construction

[0015] The first step of the present invention is to construct a recombinant expression vector carrying the antithrombin III (ATIII) gene. The vector construction method can be carried out with reference to the description in the patent specification with application number 2011102521955. The specific steps are as follows:

[0016] 1. Construction of the recombinant vector containing the target gene rhATIII

[0017] Total RNA was extracted from human liver tissue, and the first strand of cDNA was synthesized with oligo(dT) as primer. Using the first strand of the synthesized cDNA as a template, PCR primers were designed according to the published ATIII gene sequence (GenBank, D29832), and a unique Xho1 restriction site was introduced at the 5' end of the primer for PCR amplification, and the PCR product was It was cloned into the pUC19 vector, and the ATIII sequence contained in the vector was confirmed to be consistent with the published ATIII sequence by DNA sequencing analy...

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Abstract

The invention relates to a recombinant goat fetal fibroblast for expressing an antithrombin III. A rhATIII modified fibroblast SM1sCh-AT is preserved in a China Center for Type Culture Collection (CCTCC) on August 15, 2012, and the preservation number is CCTCC C 2012103. By means of a structured rhATIII mammary gland specific expression vector, a target gene rhATIII is integrated on genomes of the goat fetal fibroblast. Specific efficient expression of the target gene in goat mammary gland tissues can be guided by cloning 5' and 3' regulation areas of a goat beta casein gene at two ends of the rhATIII gene. The recombinant goat fetal fibroblast containing the target gene rhATIII can be used as a nucleus donor cell for the structuring of transgenic cloning embryos through nuclear transfer, transgenic cloning embryos are obtained through a somatic cell nuclear transfer method, obtained embryos are transferred into receptor goat uteri, and accordingly, rhATIII modified milk goat bioreactor can be produced efficiently.

Description

technical field [0001] The invention belongs to the technical field of transgenic cloning cells, and in particular relates to a recombinant sheep fetal fibroblast expressing antithrombin III. Background technique [0002] Antithrombin III (ATIII for short) is a serine protease inhibitor and an important anticoagulant factor in human plasma. It bears 60% to 70% of the anticoagulant activity in plasma. Plays an important role in physiological blood coagulation and anticoagulation. The synthesis site of antithrombin III is the liver, which mainly inactivates thrombin by combining with thrombin Xa and XIa, thereby exerting the anticoagulant effect. Decreased levels of antithrombin III in humans are common in the following cases: [0003] a) hereditary antithrombin III deficiency; [0004] b) Acquired antithrombin III deficiency, seen in various liver diseases such as liver cirrhosis and advanced liver cancer; [0005] c) Increased loss of antithrombin III, seen in renal dise...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A01K67/027C12R1/91
Inventor 邹贤刚赵雅琳袁三平
Owner QINGDAO SAMUELS INDAL & COMML
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