人可溶性TNF受体(sTNFRI)在大肠杆菌中的表达及其活性鉴定

The expression and activity of human soluble TNF receptor I by E.coli and appraisement of its activity

目的 :构建人可溶性TNF受体I(sTNFRI)基因表达载体 ,并在大肠杆菌中高效表达。方法 :用RT PCR扩增编码人TNFRI胞外段基因片段 ,将其插入到表达载体pET 2 8a ,并转入大肠杆菌进行表达。结果 :在IPTG诱导下 ,转入外源基因的大肠杆菌BL 2 1可高效表达sTNFRI蛋白 ,SDS PAGE显示在 2 7kD处有一特异表达条带 ,其表达量占菌体蛋白总量的 31%。纯化的sTNFRI可有效封闭TNF对L92 9细胞的胞毒效应 ;间接免疫荧光显示它可特异性抑制TNF与靶细胞TNFR的结合。结论 :通过基因工程技术获得了人sTNFRI重组蛋白。

Objective:To construct and express the recombinant of human soluble TNF receptor I by E coli Methods:The cDNA coded for extracellular region of human TNFRI was amplified by RT PCR and inserted into a expression vector, pET 28a Then, the recombinant sTNFRI/pET 28a was transfected and expressed in E coli Results:After the stimulation with IPTG, sTNFRI fusion protein was effectively produced in E coli BL 21 transfected with the exogenous gene A unique band was found at 27 kD by SDS PAGE and its expression was about 31% of total protein in E coli The purified sTNFRI fusion protein was shown to be able to suppress the cytotoxicity of TNF on L929 cells and found by indirect immune fluorescence to inhibit specifically the binding of TNF with TNFR on target cells Conclusion:The recombinant human soluble TNFR I have been obtained by using the genetic engineering technology