OverlapPCR克隆黄牛脂肪特异性磷脂酶A2基因(AdPLA)及其原核表达  

Cloning of Cattle Adipose-specific phospholipase A2 Gene(AdPLA) by Overlap PCR and Its Prokaryotic Expression

作　　者：朱金龙[1] 孙晓梅[1] 张亚[1] 蓝贤勇[1] 雷初朝[1] 陈宏[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《农业生物技术学报》2011年第6期1051-1055,共5页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.30972080);国家支撑计划(No.2008ADB2B03-19);国家高技术研究发展计划(863)(No.2008AA101010);国家转基因重大专项(No.2009ZX08009-157B,2008ZX08007-002,2009ZX08007-005B-07);农业部现代肉牛产业技术体系专项(No.Nycytx-38)共同资助

摘　　要：脂肪特异性磷脂酶A2基因(adipose-specific phospholipase A2,AdPLA)在脂肪组织甘油三酯水解过程中发挥着重要的调节作用,为了构建黄牛AdPLA基因原核表达系统,本研究通过Overlap PCR方法从黄牛(Bos taurus)基因组DNA中得到了AdPLA基因编码区全长,将其克隆到pGM-T载体上,阳性克隆经测序表明,与NCBI公布的序列(GenBank No.NM_001075280)完全一致。阳性质粒经BamHⅠ和HindⅢ双酶切后,将其定向重组到pET28a(+)原核表达载体上,转化感受态大肠杆菌(Escherichia coli)BL21(DE3)中,用IPTG诱导融合蛋白表达。SDS-PAGE电泳表明,融合蛋白His-AdPLA在大肠杆菌中表达,证明成功构建了AdPLA基因原核表达系统,为进一步研究黄牛AdPLA基因的功能和AdPLA蛋白产品开发提供了依据。Adipose-specific phospholipase A2 gene（AdPLA） is a major regulator of adipocyte lipolysis,and in order to construct the prokaryotic expression system of cattle AdPLA gene,the coding sequence of AdPLA gene was obtained by Overlap PCR method from cattle（Bos taurus） genomic DNA and cloned into pGM-T vector,the positive clone sequencing result was same as the sequence in GenBank（GenBank No.NM_001075280）.The positive plasmid was digested with BamHⅠ and HindⅢ,then the fragment was ligated with the expression vector pET28a（＋）,the recombined plasmid was transformed into Escherichia coli BL21（DE3） and induced with IPTG.SDS-PAGE result showed that the fusion protein His-AdPLA was expressed in E.coli,which indicated the prokaryotic expression system of recombined vector pET28a（＋）-AdPLA was constructed successfully.This study provides a good foundation for further research of cattle AdPLA gene and lays a pathway for developing the AdPLA protein product in the future.

关 键 词：黄牛 脂肪特异性磷脂酶A2基因(AdPLA) 原核表达 OverlapPCR 

分 类 号：Q786[生物学—分子生物学]