农业生物技术学报2011，Vol.19Issue(6)：1051-1055,5.DOI:10.3969/j.issn.1674-7968.2011.06.010
Overlap PCR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
摘要
Abstract
Adipose-specific phospholipase A2 gene (A dPLA ) is a major regulator of adipocyte lipolysis, and in order to construct the prokaryotic expression system of cattle AdPLA gene, the coding sequence of A dPLA 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. NMOO1075280). The positive plasmid was digested with BamH. I and HindW, 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 Ris-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/Overlap PCRKey words
Cattle/ Adipose-specific phospholipase A2(AdPLA)/ Prokaryotic expression/ Overlap PCR引用本文复制引用
朱金龙,孙晓梅,张亚,蓝贤勇,雷初朝,陈宏..Overlap PCR克隆黄牛脂肪特异性磷脂酶A2基因(AdPLA)及其原核表达[J].农业生物技术学报,2011,19(6):1051-1055,5.基金项目
本研究由国家自然科学基金(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)和农业部现代肉牛产业技术体系专项(No.Nycytx-38)共同资助 （No.30972080）
