TRIM5α突变体抗HIV-1活性的研究

发布时间：2018-10-05 06:23

【摘要】：TRIM5α作为一种抑制HIV-1复制的细胞因子，首先在恒河猴细胞中被发现。TRIM5α是TRIM5基因编码的六种亚型中的一种，它的结构包括RING结构域、B-box2结构域、Coiled-coil结构域以及其他TRIM5亚型中不具备的位于羧基端的B30.2结构域。病毒外膜与宿主细胞膜融合进入细胞后，病毒衣壳有序解体释放核酸之前，TRIM5α蛋白多聚体可以与病毒衣壳特异性地结合。TRIM5α具有的泛素连接酶活性，使衣壳通过泛素依赖的蛋白酶体途径被降解，病毒衣壳的不成熟解体阻断了病毒的复制周期，从而达到抑制病毒的目的。TRIM5α蛋白的抗反转录病毒活性具有鲜明的种属特异性，与其B30.2域中特定的氨基酸有关。本课题采用重叠PCR定点突变技术，将人的野生型TRIM5α基因B30.2区域的第332位精氨酸突变为亮氨酸、丝氨酸和谷氨酰胺，，构建出点突变的TRIM5α真核表达载体。另外将人的野生型TRIM5α进行片段替换，构建出含有TFP基序的TRIM5α真核表达载体。亚细胞定位研究显示野生型及突变型GFP-TRIM5α融合蛋白主要分布在胞浆中，过表达时出现点状聚集的现象。突变位点的存在不影响蛋白质的定位。瞬时表达TRIM5α突变体，同时包含TFP基序和R332P点突变的小片段TRIM5αRHE突变体具有较高的抗病毒活性，突变体R332Q和RH的作用次之，三者的抗HIV-1活性均高于R332P。
[Abstract]:As a cytokine that inhibits HIV-1 replication, TRIM5 伪 was first found in rhesus monkey cells as one of the six subtypes encoded by TRIM5 gene. Its structure includes the RING domain, B-box2 domain, Coiled-coil domain, and the B30.2 domain at the carboxyl terminal, which is not found in other TRIM5 subtypes. After fusion of virus outer membrane and host cell membrane into cells, virus capsid disintegrates and releases nucleic acid in an orderly manner, and TRIM5 伪 protein polymer can specifically bind to virus capsid. TRIM5 伪 has ubiquitin ligase activity. The capsid was degraded through the ubiquitin dependent proteasome pathway, and the premature disintegration of the virus capsid blocked the replication cycle of the virus. Thus, the antiretrovirus activity of TRIM5 伪 protein had a distinct species specificity. It is related to the specific amino acids in the B30.2 domain. In this study, a point mutant eukaryotic expression vector of TRIM5 伪 was constructed by mutating the 332nd arginine of human wild-type TRIM5 伪 gene B30.2 into leucine, serine and glutamine by using overlapping PCR site-directed mutation technique. In addition, TRIM5 伪 eukaryotic expression vector containing TFP motif was constructed by replacing human wild-type TRIM5 伪 fragment. Subcellular localization showed that wild-type and mutant GFP-TRIM5 伪 fusion proteins were mainly distributed in the cytoplasm. The presence of mutation sites does not affect the localization of proteins. The short fragment TRIM5 伪 RHE with TFP motif and R332P point mutation had higher antiviral activity, followed by R332Q and RH. The anti-HIV-1 activity of the three mutants was higher than that of R332P.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.91

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