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Experimental study of the vif gene mutation analysis of HIV-1 isolates and Vif protein function

Author: ZhangYunZhi
Tutor: LuHongZhou；ChengXunJia；JiangWeiMin
School: Fudan University
Course: Internal Medicine
Keywords: HIV Vif APOBEC3G Mutation Polyclonal antibody Cell model
CLC: R346
Type: Master's thesis
Year: 2008
Downloads: 53
Quote: 0
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Abstract


Objective: In this study, HIV-1 isolates vif gene and protein structure -depth understanding ; vitro recombinant of HIV-1Vif protein preparation Vif mouse polyclonal antibodies and build the Vif lentiviral vector infection cell model for further study of the use of RNAi technology interfering with Vif expression , demonstrate the feasibility of gene therapy for intervention in HIV-1 Vif start . Methods: vif genes Shanghai HIV-1 isolates was amplified by RT-PCR in vitro vif gene mutation rate in Shanghai HIV-1 isolates by sequencing and comparative analysis with international standard HIV-1 isolates , and export of amino acid variation ; the vif genes pET32b ( ) to further build the prokaryotic expression vector , expressed and purified Vif protein cell lines BL21 star (DE3) (pLysS) prepare mouse polyclonal antibody to mice immunized with purified Vif protein by ELISA restructuring Vif protein and its polyclonal antibody function ; build HIV-1Vif lentiviral expression vector , infected HEK293T cells and normal human PBMC cells , and Real-Time PCR method and SDS-PAGE analysis vif gene and Vif protein expression . Results: The study data is confirmed by the Shanghai isolates of HIV-1 vif gene and the international standard of HIV strains there is a higher mutation rate , but there are relatively conserved nucleotide sequence of between 150bp ~ 245bp (579bp) , VIF protein amino acid there 's variation , whether these amino acid variation sites affect the function of Vif is not very clear and needs further study : the restructuring of the research , the of HIV-1Vif protein expression , and successful preparation of its polyclonal antibody and confirmed by experiment serum samples of patients with AIDS does not exist or there is only a small amount of the HIV-1Vif antibody can also be detected in HIV-1 infected individuals value further study ; this study successfully constructed lentiviral vector expressing Vif protein infected normal human PBMC cell model and HEK293T cells model . Conclusion : the HIV-1Vif - based importance in the pathogenesis of HIV-1 , in-depth study of its gene and protein able to find new means of HIV infection / AIDS gene therapy , the study of HIV-1vif gene sequence , find relatively conservative sequence of the target sequence selection for RNAi role ; the prepared of the Vif mouse polyclonal antibody can be used for the detection of Vif protein ; build Vif interfere with cell model can be used for further in-depth study .

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