人热休克蛋白70基因的克隆、表达及鉴定

Cloning and Expression of Human Heat Shock Protein 70 Gene and Identification of Expressed Product

目的克隆人热休克蛋白70(HSP70)基因,构建其原核高效表达载体。方法将PCR扩增的HSP70基因克隆到原核高效表达载体pET-28b中,转化大肠杆菌JM109。挑选阳性克隆,提取质粒pE28b70F,转化大肠杆菌BL21(DE3),IPTG诱导表达目标蛋白。结果在大肠杆菌中成功表达了HSP70,表达量约占细菌总蛋白的22·3%,其中可溶性目标蛋白占上清总蛋白的12%。Westernblot表明该目标蛋白具有与鼠抗人HSP70单抗特异结合的抗原活性。通过Ni2+-NTA亲和柱,从1L诱导产物中纯化出约1520mg重组蛋白,纯度在80%以上。结论已成功表达HSP70,为进一步研究其生物学功能和作用机制奠定了基础。

Objective To clone human heat shock protein 70（HSP70） gene for the construction of a prokaryotic expression vector. Methods Amplify HSP70 gene by PCR, clone into prokaryotic expression vector pET-28b, then transform to E. coli JM109. Screen the positive clones,extract plasmid pE28b70F and transform to E. coli BI21 （ DE3 ） for expression under induction of IPTG. Retraits The expressed HSP70 contained 22. 3 % of total somatic protein, of which 12% were soluble protein. Western blot showed specific reaction of the expressed protein with mouse anti-human HSP70 McAb. About 15-20 mg of recombinant protein was purified from 1 L of culture supernatant of recombinant E. coli by Ni^2＋ -NTA affinity column chromatography, and reached a purity of more than 80%. Conclusion HSP70 was successfully expressed,which laid a foundation of further study on its biological function and action mechanism.