人源热休克蛋白HSC70与HPV16型E7融合蛋白在大肠杆菌中的表达、纯化和活性检测

Expression of fusion protein of Human HSC70 and HPV-16 E7 in E.coli and its purification and biological assay

目的克隆人热休克蛋白HSC70的N端具有ATPase活性的结构域(简称为A基因)与HPV16型E7的融合基因,在大肠杆菌中表达,并纯化获得有生物活性的AE7融合蛋白。方法利用PCR方法扩增获得A基因片段,插入原核表达载体pET29b中,再通过PCR方法扩增HPV16型E7基因片段,插入A基因的3′端,构建原核表达质粒pET29b-AE7,并转化大肠杆菌BL21(DE3),表达产物经离子交换层析和金属螯合层析纯化后,在一定的剂量下皮下免疫小鼠,检测对HPV-16的E7基因感染的预防及治疗作用。结果重组质粒pET29b-AE7经DNA序列测定确认。SDS-PAGE检测,表达产物分子量为66kD,大部分为包涵体。纯化后产物经SDS-PAGE电泳分析纯度>90%。纯化产物AE7在一定的剂量下皮下免疫小鼠,对HPV-16的E7基因的感染有预防及治疗作用。结论AE7融合蛋白的获得为进一步临床研究奠定基础。

Objective To clone and express AE7 fusion gene in E. coli BL21 （DE3） and purify AE7 fusion protein with bioactivity. Methods N terminal fragment （ATPase activity region） of Human HSC70 （abbreviated as A gene）and HPV type 16 E7 gene were prepared by PCR. The two genes were cloned into the expression vector pE129b to construct fusion gene expression plasmid pET29b-AE7 and transformed into E. coli BL21 （DE3）. The expressed product was purified by anion exchange chromatography and metal ion chelate chromatography and the purified product was injected subcutaneous into C57BL/6 mice for evaluating its roles in preventing and treating HPV 16-related ncoplasia. Results The recombinant plasmid pET29b-AE7 was confirmed by DNA sequencing. The molecular weight of expressed AE7 fusion protein was 66kD determined by SDS-PAGE. Most of the recombinant protein existed as insoluble inclusion body. The purity of the product was over 90% analyzed by SDS-PAGE. Finally, AE7 immunization can effectively prevent and treat HPV 16-related neoplasia in mice. Condusion The development of AE7 fusion protein lays a foundation of future study on its clinical application.