【摘要】 目的克隆sCD40L功能性片段(E107L261)基因并在大肠杆菌体系进行表达。方法从CD40L胞外段全长基因通过PCR方法扩增出sCD40L功能性片段(E107L261)的基因,并在其N端融合6个组氨酸(His);经PCR、酶以及DNA测序证实;将融合得到的sCD40L基因插入pET30a表达载体,转化大肠杆菌BL21(DE3)表达体系进行诱导表达。结果构建了pET30asCD40L原核表达载体;将转化菌BL21(DE3)诱导表达后经SDSPAGE电泳以及WesternBlotting鉴定显示在18kd处有sCD40L功能性片段(E107L261)的高效表达。结论sCD40L功能性片段基因的克隆及其表达为进一步研制其同源三聚体形式的功能性重组蛋白奠定了基础。更多还原

【Abstract】 Objective To clone the functional fragment of sCD40L(E107-L261), and express the protein in Escherichia coli.Methods The functional fragment of sCD40L gene(E107-L261) was amplified by PCR from the extracellular gene of CD40L. In this study, the gene coding hexahistidine was fused at N-terminal of sCD40L gene coding E107-L261. The fusion gene was amplified and cloned into expression plasmid pET30a. The protein sCD40L with His-tag at the N-terminal was expressed in E.coli BL21 (DE3) after being induced by IPTG.Results The bacterial cell expression vector pET30a-sCD40L was constructed and transformed into the expression E.coli, BL21. Induced BL21 was able to express highly the objective protein as inclusion bodies form. It was confirmed by SDS-PAGE and Western Blotting. Its Mr was about 18kd.Conclusion Cloning of functional fragment of sCD40L gene and expression of the protein provide a sound basis for producing the recombinant trimeric protein of CD40L.更多还原