HPV6型E6与结核杆菌HSP70融合基因的构建及原核表达

目的构建HPV6型E6抗原与结核杆菌HSP70的融合基因,在大肠杆菌中进行表达和纯化。方法利用PCR方法扩增结核杆菌HSP70基因,插入原核表达载体pGEX-4T-1;经测序正确后,再通过PCR方法扩增HPV6型E6基因片段,测序后插入HSP70基因的5′端,构建HPV6E6与结核杆菌HSP70的N端融合基因原核表达质粒pGEX-E6-HSP70;在含有该质粒的大肠杆菌DH5α,进行诱导表达和分离纯化。结果成功地构建了pGEX-E6-HSP70原核表达质粒,诱导表达产物与抗GST抗体在113 kD处有很强的交叉反应;大肠杆菌超声破碎后的上清液用SDS-PAGE检测到表达的蛋白多为可溶性融合蛋白;经GST融合蛋白表达系统纯化,得到了E6与结核杆菌HSP70的可溶性融合蛋白。结论成功获得HPV6型E6与结核杆菌HSP70的融合蛋白,为研究HPV6型E6与结核杆菌HSP70融合蛋白疫苗在尖锐湿疣治疗中的作用提供了材料。

Construction and Prokaryotic Expression of Fusion Gene E6 and Mycobacterium Tuberculosis HSP70

Objective To construct fusion gene E6 and mycobacterium tuberculosis HSP70.The fusion gene was expressed in E.coli.and the fusion protein was purified.Methods The E6 and HSP70 gene were amplified by PCR and were cloned into expression vector pGEX-4T-1 after being sequenced.The DH5α contained this expression vector was induced by IPTG.The fusion protein was purified with GSTrap FF column.Results The E6 and HSP70 gene were amplified successfully and their sequence were identical to the reported in the GeneBank.The fusion gene PGEX-E6-HSP70 was constructed successfully.A GST fusion protein of 113 kD was detected with anti GST antibody by Western blotting.The SDS-PAGE showed that most of recombinant protein was soluble.Conclusion The fusion protein E6 and HSP70 was expressed and purified successfully.Which prepare the materials for further research of the fusion protein as a vaccine.