抗菌肽-X基因的克隆及在大肠杆菌中的表达

Cloning of cecropin X gene and its expression in E.coli

用PCR技术获得抗菌肽-X基因、TNFα基因,与温度诱导的表达载体pRC连接成为重组表达载体,导入大肠杆菌TG1,通过温度诱导表达重组蛋白。将重组质粒转入不同的表达菌中进行表达,经SDS-PAGE选出E.coliBL21(DE3)为最佳表达的宿主菌。培养后,离心得菌体,经超声破碎离心得包涵体,溶解后用CNBr切割并透析,最后经CM52纤维素柱分离纯化得到有活性高纯度的抗菌肽-X。

Cecropin X gene and TNFαgene were amplified by PCR,then cecropin X gene was fused to the3'-end of the TNFαgene and the fusion gene was directly under the control of an temperature inducible promoter in pRC.This fusion gene was overexpressed in E.coli TG1.Assayed by SDS-PAGE,choosing the E.coli BL21(DE3)as the host cell for expression of plasmids.After cultivating,the cells were harvested and sonicated and centrifuged to ob tain the inclusion body which was solved and then cleaved by CNBr.Finally,get high-purified cecropin X with an ti-bacterial activity by one-step cellulose CM52column purification.