死亡素在大肠杆菌中的融合表达

Fusion expressing of thanatin in escherichia coli

目的:构建死亡素基因重组表达载体,并以融合蛋白的形式在大肠杆菌中诱导表达。方法:人工合成死亡素基因片段,克隆于质粒pUC18,转化大肠杆菌DH5α,挑选阳性菌落,提取重组质粒双酶切,电泳回收目的基因,与经相同酶切的pGEx-4T-l连接构建重组表达载体,转化大肠杆菌BL21(DE3),筛选阳性菌落,抽提质粒进行鉴定,测序正确的重组质粒转化大肠杆菌BL21,IPTG诱导表达。结果:重组表达质粒经双酶切、PCR及测序鉴定,证明目的基因正确插入。IPTG诱导表达后,SDS-PAGE显示出现目的条带,与预期结果一致。结论:成功构建了死亡素基因的原核表达载体,并诱导表达出目的融合蛋白。

Objective To construct the recombinant expressing plasmid of thanatin and induce its expression in escherichia coli as fusion protein.Method The synthesised DNA of thanatin was cloned into pUC18 and transformed into E.coli DH5α.The positive clony was chose and the plasmid was extracted for identification.The correct recombinant plasmids proved by sequencing were transformed into E.coli BL21 （DE3）,inducing with IPTG.Results The target gene were correctly inserted into the recombinant expressing plasmids identified by restriction analysis,PCR and sequencing.Induced under IPTG,the target band appeared as expectlly on SDS-PAGE profile.Conclusion The prokaryotic expressing plasmid of thanatin is constructed successfully,and the target fusion protein is expressed.