重组小鼠树突状细胞因子DCF1的原核表达和纯化

Prokaryotic Expression and Purification of Mouse Recombinant Dendritic Cell-derived Factor 1-DCF1

目的:克隆小鼠重组树突状细胞因子DCF1蛋白进行原核表达、纯化与鉴定。方法:采用PCR从小鼠脑cDNA克隆dcf1基因,构建DCF1原核表达重组质粒(pET30a-DCF1)并转化E.coli的BL21(DE3)菌株。IPTG诱导重组蛋白表达,并在变性条件下经Ni sepharose FF6亲和层析柱纯化,再通过SDS-PAGE和Western blotting鉴定。结果:成功克隆到大小为972bp的小鼠源dcf1基因片段并准确插入表达载体pET30a,0.1 mmol/L IPTG诱导转化菌2h可表达大量的DCF1蛋白,并可经Ni Sepharose FF6柱亲和层析得到高度纯化。结论:成功获得纯化的42kDa重组小鼠DCF1蛋白,为后续进行DCF1蛋白功能研究奠定了基础。

Objective：To clone,express and purify mouse recombinant dendritic cell-derived factor 1-DCF1.Method：dcf1 gene was amplified from mouse brain cDNA by PCR,and subcloned into prokaryotic expression vector pET30a.This recombinant plasmid was transformed into E.coli BL21（DE3）.Recombinant DCF1 expression was induced by IPTG.Resulted protein was purified by Ni sepharose ff6 resin under denature condition.SDS-PAGE and Western blot method were used to identify the expression and purification of recombinant DCF1.Result：Mouse dcf1 cDNA fragment of 972bp was obtained,and was subcloned into prokaryotic expression vector pET30a.A 42kDa recombinant DCF1 was successfully induced to express in E.coli and further purified by Ni sepharose FF6 affinity chromatograph.Conclusion：A method for prokaryotic expression and purification of mouse recombinant DCF1 was successfully established.This method laid a foundation for the subsequent DCF1 research.