脂联素原核表达载体PQE30/ADPN的构建及其表达与纯化

目的构建大鼠全长脂联素(fAd)和球状域脂联素(gAd)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,对表达产物进行纯化和鉴定。方法将纯化的脂联素克隆产物与原核表达载体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M15感受态细胞中,并用IPTG诱导表达,并通过亲和层析、去盐、去除内毒素等纯化蛋白。结果PCR获得长度分别为684bp(fAd)和402bp(gAd)的目的片段,经PQE30原核表达载体连接、筛选及序列分析后,证实所插入目的片段与GenBank中脂联素序列(序列号:NM_144744)完全一致;含重组脂联素质粒的大肠杆菌在30℃经0.5mmol.L-1IPTG诱导6h时,可溶性蛋白表达量最高。结论成功克隆大鼠脂联素基因,并在大肠杆菌中获得有效表达。

Construction of procaryon PQE30/adiponectin expression vector and its expression and purification

Objective To construct the PQE30/adiponectin(ADPN)vector and identify its expression in Escherichia(E.)coli.Methods Using enzymes to digest plasmids,the encoding fragment of rat ADPN gene was ligated into expression vector PQE30.After transformation,it was introduced into E.coil M15 competent cells.The recombinant vector PQE30/ADPN was constructed,and its expression was induced by IPTG.The protein was purified by affinity chromatography,and desalting to remove endotoxin.Results 684 bp(fAd)and 402 bp(gAd)fragments were obtained by PCR clone.After blotting and sequencing,the results were matched to the cDNA of rat ADPN,whose accession in GenBank was NM_144744.An expression was obtained by 0.5 mmol·L-1 IPTG inducement in E.coil M15.Conclusion The procaryon PQE30/adiponectin expression vector was successfully constructed and expressed.