【摘要】 目的:构建蛇毒锯鳞蝰素(Echistatin,Ecs)多顺反子串联多拷贝基因。方法:以pMD18T-Ecs为模板,利用三对引物分别扩增Ecs基因,每个Ecs基因都有独立的起始和终止密码子,然后通过三个Ecs基因之间合适的酶切位点使之串联,再与表达载体pET30a连接后得到三拷贝重组质粒,在三个Ecs基因之间分别有SD序列和SD间隔序列。将质粒转化E.coliBL21(DE3)后IPTG诱导表达,18%SDS-PAGE和Westernblot鉴定结果。结果:Ecs的表达量占全菌总蛋白的18%,实现了Ecs的串联表达。结论:Ecs多顺反子的串联表达为小分子蛋白的体外制备提供了一种全新的思路和方法。更多还原

【Abstract】 Objective: To construct a polycistron tandem repeated Echistatin (Ecs) gene. Methods: Three Ecs genes with independent initiation and termination codon were ligated tandem through restriction enzyme sites after amplified with 3 pairs of primers using pMD18T-Ecs as template. The polycistron Ecs gene was inserted into pET30a and expressed in E.coli BL21(DE3) with IPTG induction. The expression results were identified by 18% SDS-PAGE and Western blot. Results: The expression of Ecs polycistron was accomplished with 18% expression level of total protein determined by SDS-PAGE and Western blot. Conclusion: The successful expression of Ecs polycistron provided a new method for the preparation of low molecular weight protein.更多还原