山羊c-Myc原癌基因克隆、原核表达和GST-Myc融合蛋白纯化  

Cloning and Prokaryotic Expression of Goat c-Myc Proto-oncogene,and Purification of GST-Myc Fusion Protein

作　　者：赵华[1] 杨照海[1] 刘平[1] 卢晟盛[1] 卢克焕[1] 毕方方[1] 郑喜邦[1] 

机构地区：[1]广西大学动物科技学院,南宁530005

出　　处：《农业生物技术学报》2010年第5期931-937,共7页Journal of Agricultural Biotechnology

基　　金：广西区自然科学基金项目(桂科自No.0728019;桂科自No.0991043);广西亚热带生物资源保护利用重点实验室开放课题(No.SB0907);广西大学科研基金项目(No.X081102)共同资助

摘　　要：本研究的目的是通过分子克隆、原核表达和蛋白纯化技术获得山羊GST-Sox2融合蛋白。用RT-PCR方法从山羊(Capra hircus)肠组织克隆了c-Myc基因,通过TA克隆,构建了pMD18-T-Myc质粒。DNA测序证明,山羊c-Myc全长cDNA约1320bp,该序列包含一个完整的开放阅读框,编码439个氨基酸,经分析该序列与绵羊(Ovisaries)的c-Myc序列同源性为99.2%。将该cDNA片段亚克隆至pGEX-KG载体,构建了重组质粒pGEX-KG-Myc。经酶切鉴定和测序,将重组质粒导入大肠杆菌(Escherichia coli)BL21,由IPTG诱导表达,以谷胱甘肽-Sepharose4B亲和纯化GST-Myc融合蛋白。SDS-PAGE分析表明,纯化的GST-Myc融合蛋白约75kD,与预期值相符,且呈现清晰的单一条带。Western blot检测证实,该融合蛋白能够被GST抗体所识别。本实验克隆了山羊c-Myc基因,获得了高纯度的GST-Myc融合蛋白,为其多克隆或单克隆抗体的制备提供了基础资料,为山羊iPS细胞(induced pluripotent stem cells)检测创造了条件。This paper was to obtain goat（Capra hircus） GST-Myc fusion protein by means of molecular cloning,prokaryotic expression and protein purification.The c-Myc cDNA of goat was amplified by RT-PCR from intestine tissues,and plasmid pMD18-T-Myc was constructed by T-A cloning.DNA sequencing showed that the cDNA was 1 320 bp,with a complete open reading frame that encoded 439 amino acids,sharing higher nucleotide homology（99.2%） with that of sheep（Ovis aries）.The cDNA fragment was subcloned to pGEX-KG,and recombinant plasmid pGEX-KG-Myc was constructed,which was verified by restriction endonuclease analysis and DNA sequencing.The recombinant plasmid was transformed into E.coli BL21,and GST-Myc fusion protein was expressed with induction of IPTG,and then purified with Glutathione-Sepharose 4B argrose.Illustrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）,the purified fusion protein was about 75 kD,a clear,and single band,which was expected.Western blot assay revealed that fusion protein could be identified by GST antibody.In conclution,we have cloned goat c-Myc gene,obtained GST-Myc fusion protein with higher purity,which will lead to preparation of polyclonal or monoclonal anti-Sox2 antibody,and further to application in identification of goat iPS cells（induced pluripotent stem cells）.

关 键 词：山羊 C-MYC基因 分子克隆 原核表达 蛋白纯化 

分 类 号：S513.01[农业科学—作物学]