干扰素诱导蛋白IFI16与HPV16E6、E7蛋白相互作用的研究

Interaction between interferon-inducible IFI16 protein and human papillomavirus protein E6 and E7

目的:研究人乳头瘤病毒(humanpapillomavirus熏HPV)16型E6、E7蛋白与干扰素诱导蛋白IFI16的相互作用,初步揭示IFI16拮抗HPV16E6、E7致瘤活性的分子机制。方法:(1)体外GST阻滞分析:克隆、表达及纯化GST鄄HPV16E6和GST鄄HPV16E7融合蛋白,将含有IFI16蛋白的EB病毒阴性伯基特淋巴瘤穴BJAB雪细胞提取物与上述纯化蛋白孵育,通过WesternBlotting分析蛋白质的体外相互作用;(2)免疫共沉淀分析:构建表达His鄄HPV16E6蛋白的真核表达载体,将表达有IFI16和His鄄HPV16E6蛋白的293T细胞裂解物与抗His抗体孵育,用IFI16抗体检测共沉淀蛋白复合物中IFI16蛋白的存在;将表达有IFI16和HPV16E7蛋白的CaSki和SiHa细胞裂解物与HPV16E7抗体孵育,检测共沉淀蛋白复合物中E7与IFI16是否结合。结果:成功构建了原核表达质粒pGEX鄄4T/HPV16E6及pGEX鄄4T/HPV16E7,并表达、纯化获得GST鄄HPV16E6及GST鄄HPV16E7融合蛋白。体外GST阻滞分析结果表明,用IFI16单抗做WesternBlotting分析可检测到复合物中IFI16蛋白,表明GST鄄HPV16E6和GST鄄HPV16E7均可与IFI16蛋白结合。免疫共沉淀分析进一步证实,共沉淀蛋白复合物中也可检测到IFI16蛋白,证明在细胞中HPV16E6和E7蛋白也能与IFI16蛋白结合。结论:无论在体外还是在细胞中,抑瘤因子IFI16蛋白均能特?

Objective: To study the molecular mechanism underlying that interferon-inducible IFI16 protein overcomes the tumorgenesis activities of HPV16E6 and E7, the authors investigated whether HPV16E6 and E7 could associate with IFI16. Methods: GST-Pulldown was performed to examine the interaction between IFI16 and HPV16E6/E7 in vitro. Bacteria expression plasmids expressing GST-E6 and GST-E7 were generated and GST fusion proteins purified. The purified GST-E6 and GST-E7 were incubated with the whole cell extracts prepared from BJAB cells that expressed endogenous IFI16, and the IFI16 detained by GST fusion proteins were detected using Western Blotting assay with anti-IFI16 antibodies. In vivo association of IFI16 and HPV16E6 was tested via co-immunoprecipitation (Co-IP) using transfected mammalian cells. Briefly, an expression pcDNA4HisMax/HPV16E6 plasmid expressing His-HPV16 E6 protein was constructed and used to transfect 293T cells together with another expression pRcCMV/IFI16 plasmid expressing IFI16 protein. The whole cell lysate prepared from these transfected cells were incubated with anti-His serum followed by protein A agarose and the presence of IFI16 in the immunoprecipitated protein was visualized by immunoblotting with anti-IFI16 antibodies. Furthermore, in vivo interaction between IFI16 and HPV16E7 was also revealed via a Co-IP assay with cell extracts prepared from both SiHa and Caski cells in which the endogenous IFI16 and HPV16 E7 proteins were expressed. The cell lysates were immunoprecipitated with anti-HPV16 E7 serum and protein complex was detected with anti-IFI16 antibodies. Results: The plasmids expressing GST-HPV16E7 and His-HPV16E6 were successfully generated and the fusion proteins of interests were verified to be correct. In vitro pulldown assay clearly demonstrated that both GST-HPV16E6 and GST-HPV16E7 were capable of binding to IFI16, and co-immunoprecipitation assays revealed that HPV16E6/E7 and IFI16 were present in the same protein complexes and also associated with each other in vivo. Conclusion: IFI16 associates with HPV16E6 and E7 proteins both in vitro and in vivo. These foundings could provide potential molecular targets for designing compounds to treat human cervical cancer.