人热休克蛋白70的原核表达和纯化的实验研究

Prokaryotic Expression and Purification of Human Heat Shock Protein 70

目的在大肠杆菌中表达人源性热休克蛋白70基因并纯化表达产物。方法以人HSP0 cDNA为模板,采用PCR方法进行扩增,将PCR产物克隆到表达载体pET30a上,将重组质粒pET30a-HSP70转化大肠杆菌BL21上,经IPTG诱导后,表达产物进行SDS-PAGE及Western blot验证并纯化。结果应用PCR方法扩增出约1900bp的目的片段;经酶切鉴定和DNA测序证实,HSP70基因成功地克隆到原核表达载体pET-30a上;转入重组质粒的大肠杆菌经IPTG诱导后,进行SDS-PAGE,发现在分子量约为70kD处有蛋白表达,Western blot证实其为目的蛋白,纯化后的HSP70纯度达到90%以上。结论在大肠杆菌中已成功地表达了HSP70蛋白并且经过纯化,为研究HSP70的结构、功能及临床应用提供了必要条件。

Objective To express human heat shock protein 70 （HSP70）in E. coli and purify the expressed product. Methods The cDNA fragment encoding human HSP70 was amplified by PCR. And the PCR products were then cloned into vector pET30a. The recombinant plasmid, pET30a -HSP70 was transformed to E. coli strain BL21. Under the inducement of IPTG, the products after profication were detected by SDS-PAGE and Western blot. Results Results showed that a fragment about 1900bp were amplified by PCR. The HSP70 gene was cloned into pET30a identified by enzyme digestion and DNA sequencing; Under the inducement of IPTG,a protein with Mr 72000 identified by SDS-PAGE was expressed by the E. coli B121 that contained the recombinant plasmid, and could be recognized by anti-HSP70 antibod. The purity of HSP70 after purification was more than 90%. Conclusion HSP70 was successfully expressed in E. coli and purified. The study provide a necessary condition for studying structure,function and clinic application of HSP70.