人脂联素原核表达载体PQE30/ADPN的构建及在大肠杆菌中的表达

目的:构建人脂联素重组表达载体PQE30/ADPN,并在大肠杆菌宿主系统中表达出脂联素蛋白,对表达产物进行鉴定。方法:将构建好的人脂联素克隆载体PUC57/ADPN与原核表达载体PQE30通过双酶切方法位点特异地连接在一起,再转化入大肠杆菌JM109感受态细胞中。通过筛选得到含有人脂联素基因的重组载体PQE30/ADPN,并用IPTG在大肠杆菌M15中诱导表达。结果:PCR获得长度为753bp目的片段,经PQE30原核表达载体连接、筛选及序列分析后,证实所插入的目的片段与GenBank检索的人脂联素cDNA序列(Accession NM-004797)100%匹配;含重组Adiponectin质粒的大肠杆菌经0.4mmol/L的IPTG诱导6h后有表达。结论:应用并成功构建了人脂联素原核表达载体PQE30/ADPN,且在大肠杆菌中获得有效表达。

Construction of Prokaryoti PQE30/Adiponectin Expression Vector and Its Expression in Escherichia Coli

Objective:To construct the PQE30/adiponectin(ADPN) vector and identify its expression in Escherichia(E.) coli.Methods:The encoding fragment of human ADPN gene was obtained from a recombinant plasmid PUC57/ADPN.Using enzymes to digest plasmids,the encoding fragment of human ADPN gene was ligated into expression vector PQE30.After transformation,it was introduced into E.coli JM109 competent cells.After choosing and sequencing,the recombinant vector PQE30/ADPN was constructed,and its expression was induced by IPTG.Results:A 753 bp fragment was obtained by PCR clone.After blotting and sequencing,the results were matched to the cDNA of human ADPN,which accession of the GenBank was NM-004797.An expression was obtained by 0.4 mmol/L IPTG inducement in E.coli M15.Conclusion:The prokaryoti PQE30/ADPN vector was first successfully constructed and expressed.