人可溶性肿瘤坏死因子受体1基因的克隆及原核表达

目的 构建人可溶性肿瘤坏死因子受体1基因的重组质粒，进行原核表达。方法 以Hela细胞的总RNA为模板，用RT-PCR方法扩增sTNFR1全编码区基因片段，构建含有目的片段的T载体克隆及原核表达载体pMAL-c2x重组质粒亚克隆，经异丙基-B-D半乳糖苷酶(IPTG)诱导重组质粒菌表达sTNFR1，以淀粉树脂亲和层析法纯化重组蛋白，用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)对重组蛋白进行分析。结果经核苷酸序列测序和sDS—PAGE法鉴定，成功构建了人sTNFR1重组质粒基因工程菌，并表达和纯化了sTNFR-MBP融合蛋白。结论成功地构建了人sTNFR1重组质粒克隆，表达并纯化了sTNFR1-MBP融合蛋白。

Cloning and Expression of Human sTNFR1 in E.Coli JM109

Objective To construct the recombinant plasmid carrying human sTNFR1 cDNA, and express sTNFR1 in E. Coli JM109. Methods Total RNA was extracted from Hela cells, and used as a template to amplify human sTNFR1 cDNA by RT-PCR. The PCR products were cloned into T vector, and then sTNFR1 cDNA fragment was subcloned into a prokaryotic expression plasmid pMAL-c2x. The recombinant plasmid was transferred into E. Coli JM109, and induced by IPTG to express fusion protein sTNFR1-MBP. sTNFR1-MBP was purified by amylose resin affinity chromatography(ARAC), and analyzed by SDS-PAGE. Results A 558 bp human sTNFR1 cDNA was amplified by RT-PCR, and successfully inserted into plasmid pMAL-c2x. sTNFR1-MBP was produced in E.Coli after IPTG induction, and a 66 KD sTNFR1-MBP was purified by ARAC. WTHZ]Conclusion Recombinant plasmid carrying human sTNFR1 cDNA was successfully constructed and epxressed in E. Coli JM109.