人肥胖基因大肠杆菌表达载体的构建及其诱导表达  

Construction of Expression Vector of Human Obese Gene and Its Induced Expression in Escherichia coli

作　　者：刘建忠[1] 敖敬[1] 田为宇[1] 周卫[1] 鲁礼林[1] 张晓龙[2] 

机构地区：[1]武汉科技大学化学工程与技术学院,武汉430081 [2]武汉科技大学计算机学院,武汉430081

出　　处：《华中农业大学学报》2008年第6期705-708,共4页Journal of Huazhong Agricultural University

基　　金：国家"863"项目(2001AA213081);东南大学生物电子学国家重点实验室开放课题资助

摘　　要：通过设计1对PCR引物(上游引物,5-′GCCATATGGTGCCGATCCAAAAAG-3′,下游引物,5-′GAGAATTCCCTTCAAGGCCTCAG-3′),采用PCR方法扩增出人肥胖基因编码成熟肽的核苷酸序列,将扩增产物从琼脂糖凝胶上回收后插入测序载体pMD-18-T后进行了核苷酸序列测定。将经EcoRⅠ和NdeⅠ双酶切后的扩增产物插入经EcoRⅠ和NdeⅠ双酶切后的大肠杆菌表达载体pET-28a(+),构建了可在大肠杆菌中高效表达人瘦蛋白的表达载体。将表达载体转化入大肠杆菌菌株BL21,经IPTG诱导后进行SDS-PAGE检测,结果表明构建的人肥胖基因表达载体得到高效表达,重组蛋白占到菌体总蛋白的31.2%。A pair of PCR primers were designed to amplify the encoding sequences of the mature peptide of human obese gene, the oligonucleotides were as follow-upstream primer, 5＇-GCCATATGGTGCCGATCCAAAAAG-3＇, downstream primer, 5＇-GAGAATTCCCTTCAAGGCCTCAG-3＇. The sequences were amplified by PCR and the product was collected from agarose gel, then was inserted into plasmid pMD-18-T vector for sequencing. The product of encoding sequences after double restriction endonuclease digestion with EcoR Ⅰ and NdeⅠwas inserted into plasmid pET-28a（＋） after digestion with the same two restriction endonecleases, a vector for the expression of human obese gene in E. coli was constructed. The expression vector was transformed into bacterium strain BL21 and was induced to express human leptin by IPTG, the product of expression was tested by SDS-PAGE. SDS-PAGE results showed that human obese gene was highly expressed in E. coli, the recombinant protein took 31.2% of the total bacteria proteins.

关 键 词：人肥胖基因 原核表达载体 诱导表达 

分 类 号：Q785[生物学—分子生物学] Q786