牛朊蛋白基因真核表达载体的构建及其在牛骨髓间充质干细胞中的稳定表达

›› 2012, Vol. 39 ›› Issue (7): 20-25.

牛朊蛋白基因真核表达载体的构建及其在牛骨髓间充质干细胞中的稳定表达

宋少康^1,2, 董雅娟^1,2,3, 柏学进^1,2,3, 王文明^1,2, 刘玮^1,2, 牛召珊^1,2, 赵仕全^1,2, 杨莉^1,2

1. 青岛农业大学动物胚胎工程中心,山东青岛 266109;
2. 山东省黑牛繁育工程技术研究中心,山东青岛 266109;
3. 山东布莱凯特黑牛科技股份有限公司,山东淄博 256306

收稿日期:2012-01-09 出版日期:2012-07-20 发布日期:2012-07-16
通讯作者: 董雅娟(1964－),女,山东人,教授,研究方向:黑牛繁育工程。E-mail:etcenter@126.com E-mail:etcenter@126.com
作者简介:宋少康(1986－),男,山东人,硕士生,研究方向:动物胚胎工程与转基因。
基金资助:
国家"抗病转基因牛新品种培育"项目资助(2008ZX08007-004)。

Construction and Stably Expression of Eukaryotic Expression Vector of Bovine PRNP Gene in Bovine BMSC

SONG Shao-kang^1,2, DONG Ya-juan^1,2,3, BAI Xue-jin^1,2,3, WANG Wen-ming^1,2, LIU Wei^1,2, NIU Zhao-shan^1,2, ZHAO Shi-quan^1,2, YANG Li^1,2

1. Center of Animal Embryo Engineering of Qingdao Agricultural University, Qingdao 266109,China;
2. Engineering Technology Research Center of Shandong Black Cattle Reproduction, Qingdao 266109,China;
3. Shandong Black Cattle Science & Technology Co., Ltd., Zibo 256306,China

Received:2012-01-09 Online:2012-07-20 Published:2012-07-16

摘要/Abstract

摘要： 试验构建牛朊蛋白(prion protein,PRNP)基因的真核表达载体,为进一步研究牛朊蛋白的生理功能和从细胞水平研究抗疯牛病转基因克隆牛奠定基础。采用重叠延伸PCR(splicing overlap extension PCR,SOE-PCR)法扩增获得牛PRNP基因序列,并克隆到带有DsRED2报告基因的真核表达载体pDsRED2-N1中,将双酶切、PCR、测序鉴定的阳性质粒经脂质体转染牛骨髓间充质干细胞(BMSC);通过荧光显微镜观察转染细胞,并用800 μg/mL G418对转染的细胞进行药物筛选。琼脂糖凝胶电泳显示基因合成的片段大小和构建的载体大小与预期相符;重组表达载体转染BMSC后有红色荧光出现;通过药物筛选出了稳定转染的细胞单克隆。通过SOE-PCR成功扩增了牛PRNP基因序列,并构建成真核表达载体,得到稳定表达目的蛋白的BMSC细胞。

关键词: 疯牛病; PRNP基因; SOE-PCR; BMSC

Abstract: In order to further study the physiological function of bovine prion protein and anti-mad cow disease cloned transgenic cattle basis form cellular level, we constructed a bovine prion protein gene (PRNP) eukaryotic expression vector. The PRNP gene was cloned by splicing overlap extension PCR (SQE-PCR) method, and inserted into the report with DsRED2 eukaryotic vector pDsRED2-N1.This recombinant plasmid pDsRED2-N1 was identified by digestion of endonuclease, PCR and sequencing. Then, it was transfected into bovine BMSC cell line mediated by Lipofectamine^TM2000. The transfected cells was detected by fluorescence microscopy and drug screening by 800 μg/mL G418. Electrophoresis analysis showed that the sequences were amplified and the sizes of products were accord with expectation. The recombinant plasmid was expressed in BMSC. The positive single stable transfection cell clones were also got by drug screening. The PRNP gene had been cloned by splicing overlap extension PCR method, and the recombinant eukaryotic vector stable expressed in BMSC successfully.

Key words: mad cow disease; PRNP gene; splicing overlap extension PCR; BMSC

中图分类号:

Q786

宋少康, 董雅娟, 柏学进, 王文明, 刘玮, 牛召珊, 赵仕全, 杨莉. 牛朊蛋白基因真核表达载体的构建及其在牛骨髓间充质干细胞中的稳定表达[J]. , 2012, 39(7): 20-25.

SONG Shao-kang, DONG Ya-juan, BAI Xue-jin, WANG Wen-ming, LIU Wei, NIU Zhao-shan, ZHAO Shi-quan, YANG Li. Construction and Stably Expression of Eukaryotic Expression Vector of Bovine PRNP Gene in Bovine BMSC[J]. China Animal Husbandry & Veterinary Medicine, 2012, 39(7): 20-25.

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