HPV16E7-HSP70 融合蛋白在大肠杆菌中表达及纯化

目的]在大肠杆菌中表达HPV16E7-HSP70融合蛋白并进行纯化和复性,为进一步研究HPV16E7-HSP70融合蛋白抗喉癌免疫活性奠定基础.方法]用构建的原核表达质粒pET28a HPV16E7-HSP70转化大肠杆菌BL21,异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达；通过超声波裂解细菌,高浓度尿素裂解包涵体和Ni2+离子金属螯合亲和层析纯化目的蛋白；并对融合蛋白进行复性；用SDS-PAGE及Western Blot进行分析鉴定.结果]SDS-PAGE 分析显示有分子量约为90kD的融合蛋白表达,表达产物以包涵体形式存在,表达量可达30％；纯化后目的蛋白的纯度达到95％；经Western Blot鉴定复性后的融合蛋白能与抗HPV 16E7抗体、抗HSP70抗体特异性结合.结论]HPV16E7-HSP70融合蛋白能够在体外大肠杆菌中表达获得.

Expression and Purification of the HPV16E7-HSP70 Fusion Protein in Escherichia coli

Purpose] To investigate the expression,purify and renature of HPV16E7-HSPT0 fu- sion protein in Eseherichia coli （E. coh） and to provide basis for further research. Methods] The recombinant prokaryotic expression plasmid pET28a HPV16E7-HSPT0 was transformed in E. coli BL21 and induced by isopropyl-β-D-thiogalactopyranoside （IPTG）. After the bacterial pellet was lysed by sonication, solubilizied in ultrapure urea,the fusion protein expressed in E. coli was puri- fied by Ni metal chelating chromatography following with renaturation,which was identified by SDS-PAGE and Western Blot analysis. Results] SDS-PAGE analysis showed that the fusion pro- tein with molecular weight of approximately 90 kD was expressed ,which formed inclusion body in the cytoplasm of bacteria,with the ratio of target protein to total protein of host 30%. The purity up to 95% could be achieved after purification of target protein. Western Blot showed that the pro- tein renatured could bind to HPV16E7 Ag and HSP70 Ag specifically. Conclusion] The HPVI6E7-HSP70 fusion protein could be obtained in E. coli in vitro.