Study Of The Interaction Between HIV-1 Vif Protein And Host APOBEC3G Protein In Vitro

Human Immunodeficiency Virus (HIV) is the pathogen of acquired immunodeficiency syndrome (AIDS), has severe spreading over the world in the past two decades and caused very serious harm to human life and great damage to our economic development and social progress, has already draw the universal attention of governments and societies around the globe. With further research, people have discovered that in the process of HIV infection, HIV always change host cell condition in many ways to enhance its compatibility, increases its infectivity and duplication ability. But at the same time the host cell also uses many kinds of immune defense responses to resist the HIV infection. Therefore we can say that the HIV infection is a process of the interaction between virus and the host. During this process one of the HIV coded auxiliary proteins—Virus Infectivity Factor(Vif) which interact with the host APOBEC3G protein plays a critical role in clinical progress. Studies on HIV-1 Vif protein and its interaction between the host protein APOBEC3G will be very meaningful for explaining the mechanism of HIV-1 infection and provide some ideas for new anti-HIV/AIDS strategies. We utilized the Escherichia coli system and the fission-yeast separately expressed naive recombinant APOBEC3G protein and Vif protein, consequently we confirmed the interaction between the two proteins in vitro successfully, has laid foundation for future research.Part I.HIV-1 Vif protein expression and its mouse source monoclonal antibody preparationThe Escherichia coli(E.coli) as a kind of prokaryote expression system is used commonly, not only because it's easy to culture and grow quickly, but its high expression quantity, low cost and suits for large scale culture. In addition people have already accumulated abundant experiences in utilizing the E.coli to express exogenous gene, therefore this system has been accepted generally. We subcloned HIV-1 Vif gene into plasmid pET32a then transformed it into E.coli, though induction the aim protein express high effective, after purification we obtained the Vif fusion protein, used this protein as antigen immune BALB/c mouse to prepare anti-HIV-1 Vif protein monoclonal antibody, and identified its characteristics. The main results are as follows: 1. Through RT-PCR amplification to obtain the aim gene Vif, after bi-enzymes cut subcloned into the plasmid pET32a to construct a recombinant prokaryotic expression vector, transform it into competence cell DH5α, after colony PCR and bi-enzymes cut identification, sequencing and compare with the standard sequence we obtained the express vector which has correct length and no code frame error named Vif-His-pET32a.2. Use IPTG to induce express vector Vif-His-pET32a express aim protein, we optimized the induces opportunity, the derivative density, the culture time as well as the bacteria lysis influence factors and protein elution system, discovered that the aim protein mainly exist as inclusion body.3. The induction expressed and purifies obtained the high-purity Vif fusion protein, Western the Blot show its antigenicity was good.4. Take purified Vif fusion protein as antigen to immune BALB/c mouse, has prepared 3 strains anti-HIV-1 Vif protein monoclonal antibody, after identification subtype is IgG2, the titer reaches to 16×106and has high specificity.Part II.Host APOBEC3G naive protein expression, purification and identificationThe APOBEC3G protein is a component of innate immunity, plays a very important role in the host cell natural anti-HIV-1 defense mechanism. According to past researches discovered that in T cells, APOBEC3G catalyzes deoxycytidines(dC) to deaminase lead to extensive dC→dU mutations in the minus-single-stranded viral DNA formed during HIV-1 reverse transcription. As a result in viral DNA will produce massive termination codons or causes G→A hypermutation, this might negate viral DNA synthesis and restrict viral replication. In our experiment, we subclone aim gene APOBEC3G into plasmid pET32a then transform into the E.coli system, through induction successfully expressed and purified recombinant APOBEC3G protein. The main results are as follows:1. Apply the same strategy as described above, we successfully subclone APOBEC3G gene into plasmid pET32a constructed the aim protein express vector APOBEC3G-His-pET32a.2. To induce APOBEC3G-His-pET32a express the aim protein, optimize experiment condition, we discovered that lysozyme-lysis and supersonic disintegration co-treatments can maximize the yield of naive fusion protein.3. Highly effective expressed and purified naive recombinant APOBEC3G protein, SDS-PAGE analysis and Western Blot identification shows high purity and fine antigenicity. Part III.HIV-1 Vif protein and host APOBEC3G protein interaction in vitro researchSince 2002 Ann M. Sheehy et al. discovered the CEM15 gene which has been sequenced to be the APOBEC3G gene later, for its essential function in innate immunity has draw more and more attention. The interaction between APOBEC3G and HIV-1 Vif has been a research hot spot in its field. In our research, we first expressed naive, fusion Vif protein in fission yeast(S.pombe system), and then identified its interaction with above expressed APOBEC3G protein in vitro. The main results are as follows:1. To conform and transform plasmid Vif-s-GFP into fission yeast(S.pombe system), optimized the protein express, cell-lysis and protein harvest conditions, finally prepared naive Vif fusion protein.2. Respectively use Vif-McAb and GFP-McAb carries on Western Blot to analysis the protein sample, data shows it to be the goal protein and has good antigenicity.3. Ni-NTA pull down test the interaction between APOBEC3G and HIV-1 Vif: consult the principles and the methods of GST pull down assay, for Ni-NTA Agarose is capable of absorbing 6×Histidine label protein with high specificity, takes it as the solid phase carrier to fix the bait protein APOBEC3G which would catch the prey protein Vif. Experiment result demonstrated that we have completed this interaction in vitro perfectly and found it's a kind interaction of high specificity.