哺乳动物细胞双杂交系统分析HIV-1 Vif-Cul5-SCF复合物及复合物小肽抑制剂的筛选

发布时间：2018-07-24 14:18

【摘要】：HIV-1 Vif蛋白是HIV-1病毒在体内进行复制和保持病原性所必需的,它的存在可以使病毒粒子的感染力提高10-1000倍。研究表明宿主细胞中存在一种抗病毒因子CEM15/APOBEC3G,可以调节宿主细胞的抗HIV-1活性,而这种抗病毒活性能被HIV-1 Vif所抑制。最新的研究发现Vif能与Cul 5、Rbx I、Elongin C、ElonginB等蛋白相作用,并且为其抗APOBEC3G功能所依赖。因此对于Vif抑制APOBEC3G的作用机制提出一个假设:Vif通过形成Cullin5-based SCF-like complex这样一个E3复合物,以泛素蛋白酶途径下调细胞内APOBEC3G的分子水平,使之无法有效的包装入子代病毒,从而保持了病毒的感染性。我们的实验利用哺乳动物细胞双杂交方法,对Vif-Cullin5-based E3复合物组分间的直接相互作用进行了检测,期望能确定它们的作用关系,进一步证实和完善这个作用模型。结果显示,我们测得较强的Vif与Elongin B的相互作用,而预期应测得的Vif与Elongin C的相互作用则很微弱;这与已知的文献报道不同,但与我们在进行的另一个Vif晶体研究试验结果一致。结果还表明受体蛋白Vif与泛素化靶蛋白CEM15是直接结合的,不需要通过其它连接蛋白的参与。我们还用噬菌体展示肽库对Vif蛋白进行了配体筛选实验,找到了保守序列,为Vif蛋白的小肽抑制剂的研究提供新的靶点。
[Abstract]:HIV-1 Vif protein is necessary for the replication and pathogenicity of HIV-1 virus in vivo, and its presence can increase the infectivity of virus particles by 10-1000 times. It has been shown that there is an antiviral factor, CEM15 / APOBEC3G, which can regulate the antiviral activity of host cells, which can be inhibited by HIV-1 Vif. Recent studies have found that Vif can interact with proteins such as Cul 5, Rbx, Elongin and Elongin B, and is dependent on its anti APOBEC3G function. Therefore, we propose a hypothesis that Vif inhibits APOBEC3G by forming an E3 complex such as Cullin5-based SCF-like complex, which can down-regulate the molecular level of APOBEC3G in cells through the ubiquitin protease pathway, so that it cannot be effectively packaged into the offspring virus. Thus keeping the virus infectious. In our experiment, the direct interaction between Vif-Cullin5-based E3 complex components was detected by using mammalian cell two-hybrid method. It is expected that the interaction relationship between them can be determined and the model can be further confirmed and perfected. The results show that the interaction between Vif and Elongin B is strong, but the interaction between Vif and Elongin C is very weak, which is different from the known literature. But it is consistent with another Vif crystal study we are conducting. The results also showed that the receptor protein Vif was directly bound to the ubiquitin target protein CEM15 without the involvement of other connexins. We also used phage display peptide library to screen the ligand of Vif protein and found the conserved sequence to provide a new target for the study of small peptide inhibitor of Vif protein.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392;Q789

【共引文献】