上海HIV-1分离株vif基因变异分析及Vif蛋白功能的实验研究

发布时间：2018-12-19 17:59

【摘要】： 目的:通过本研究对上海HIV-1分离株vif基因及蛋白的结构深入了解;体外重组HIV-1Vif蛋白、制备Vif的鼠多克隆抗体并构建Vif慢病毒载体感染细胞模型为进一步研究利用RNAi技术干扰Vif表达,论证以Vif入手进行HIV-1干预的基因治疗可行性。研究方法:利用RT-PCR法体外扩增上海HIV-1分离株vif基因,通过测序并与国际标准HIV-1分离株比较分析上海HIV-1分离株vif基因的突变率,并导出氨基酸变异情况;进一步构建vif基因pET32b(+)原核表达载体,利用BL21 star(DE3)(pLysS)细胞系表达并纯化Vif蛋白,以纯化的Vif蛋白免疫小鼠制备其鼠多克隆抗体,以ELISA法检测重组的Vif蛋白及其多克隆抗体的功能;构建HIV-1Vif慢病毒表达载体,感染HEK293T细胞及正常人PBMC细胞,并以Real-Time PCR法和SDS-PAGE检测vif基因和Vif蛋白的表达。结果:本研究数据证实上海HIV-1分离株vif基因与国际标准HIV毒株比较存在较高突变率,但在150bp～245bp(579bp)之间存在有相对保守的碱基序列,Vif蛋白的氨基酸存在一定的变异规律,这些氨基酸变异位点是否影响Vif的功能还不十分清楚,有待于进一步研究:本研究重组、表达的HIV-1Vif蛋白,并成功制备了其多克隆抗体,并通过实验证实AIDS患者血清标本中不存在或仅存在少量的HIV-1Vif抗体,在HIV-1感染者体内的检测价值还可以进一步研究;本研究成功构建了表达Vif蛋白的慢病毒载体感染正常人PBMC细胞模型及HEK293T细胞模型。结论:基于HIV-1Vif在HIV-1发病过程中的重要性,对其基因及蛋白的深入研究,能够找到HIV感染/AIDS基因治疗的新手段,本研究对HIV-1vif基因序列的研究,找到相对保守的序列供RNAi作用靶序列选择;制备的Vif鼠多克隆抗体可用于Vif蛋白的检测;同时构建了Vif干扰细胞模型,可用于进一步的深入研究。
[Abstract]:Objective: to understand the structure of vif gene and protein in Shanghai HIV-1 isolate. In vitro recombinant HIV-1Vif protein was used to prepare mouse polyclonal antibody against Vif and to construct Vif lentivirus vector infected cell model to further study the interference of Vif expression with RNAi technique and to demonstrate the feasibility of HIV-1 intervention with Vif. Methods: the vif gene of Shanghai HIV-1 isolate was amplified by RT-PCR method in vitro. The mutation rate of vif gene of Shanghai HIV-1 isolate was analyzed by sequencing and comparing with international standard HIV-1 isolate, and the variation of amino acid was deduced. The prokaryotic expression vector of vif gene pET32b () was further constructed, and the Vif protein was expressed and purified by BL21 star (DE3) (pLysS) cell line). The mouse polyclonal antibody was prepared by immunizing mice with purified Vif protein. The function of recombinant Vif protein and its polyclonal antibody was detected by ELISA assay. HIV-1Vif lentivirus expression vector was constructed to infect HEK293T cells and normal PBMC cells. The expression of vif gene and Vif protein was detected by Real-Time PCR and SDS-PAGE. Results: the data of this study confirmed that the vif gene of Shanghai HIV-1 isolate had a higher mutation rate than that of international standard HIV strain, but there was a relatively conserved base sequence between 150bp~245bp (579bp). The amino acids of Vif protein have a certain variation rule. It is not clear whether these amino acid mutation sites affect the function of Vif, and need to be further studied: in this study, the recombination of HIV-1Vif protein, the expression of HIV-1Vif protein, The polyclonal antibody was successfully prepared, and it was confirmed by experiments that there was no or only a small amount of HIV-1Vif antibody in the serum samples of AIDS patients. The detection value of HIV-1Vif antibody in HIV-1 infected patients could be further studied. In this study, we successfully constructed the human PBMC cell model and HEK293T cell model infected with lentivirus vector expressing Vif protein. Conclusion: based on the importance of HIV-1Vif in the pathogenesis of HIV-1, a new method of gene therapy for HIV infection / AIDS can be found by studying its gene and protein. The sequence of HIV-1vif gene was studied in this study. A relatively conservative sequence was found for the selection of target sequences by RNAi. The Vif mouse polyclonal antibody can be used for the detection of Vif protein, and the Vif interference cell model is constructed, which can be used for further study.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

【相似文献】