Constuction Of HIV-1 Vaccine Based On Recombinant Virus And Study On Antigenicity And Immunogenicity Of Their Expression Product

It Is known that (Acquired immenodeficiency yndrome) AIDS is a disease indeced by (Human immunodeficiency virus)HIV.It's expanding all over the world everday.The human being faced great challenges in researching and manufacturing related immune serum.and still can't heal AIDS because of HIV'S characteristics-it's sudden change ,double strand RNA virus attacking human immue system directly .So identifying the challenges to conducting HIV vaccine trials is more and more important. It Is now thought that an effective HIV-1 vaccine should prime both cross-neutralizing antibodies and long-lasting cytotoxic CD8+Tlymphocytes(CTLs),Previous studies show that inmunization with the recombinant virus-like particle (VLP)may induce substantial serum antibody titers and promoted both T helper cell and CTL response ,So we construct the rMVA and rFPV expressing HIV-gag-pol which can form VLP.Poxviruses have recently been sludied as potential vectors for delivery of heterologous vaccine antigen.Because these viruses abortively infect human and most mamralian cells yet still effecfivelypresent encoded forgein genes to the host inmune system Compared with the replication-compentent vaccinin virus,the MVA modified vaccinia Ankara ,all a highly attenuated vaccinia virus,lost hte ability to replicate in human and most mamralian cells except BHK-21 and CEF cells. Since the replication block of MVA in mamralian cells at a late stage morphogenesis after the synthesis of early or late paotein .SO they offer a safer but effectively alternative to other live virus vectors.We successfully finish the constrution of the recombinant shuttle vector inserting HIV-gag-pol gene pscll,which has the ability to recombine to MVA and FPV genomic DNA by using thymidine kinase(TK) gene a nonessential region in virus genome .In our study, we got rid of the factors of inhabiting expression of HIV-gag-pol gene to increase the quanlity of protein product .To confirm this attempt,We got the two rMVA transferred in HEK293 cell,The result indicate that rMVA-modified gag-pol gene show far higher levels of experssion we also make the study onantigecicity of rMVA by western -blot analysis using china HIV-1 positive serum as antibody and the result revealed that the expression product of MGP can be recogized by this antibody ,so M-PGsuggest a good antisenicity The inmunogenicity of recombinant modified vaccinia Ankara,expressing HIV-1 gag-pol (MVA-gag-pol) was investigated by priming mice with DNA and boosting with recombinant rMVA in Our study .we have detected the anti HIV-1 positive Serum of these mice by western -blot at different days .The picture of electrophogensis analysis suggest that the band of p24 antibody is most bfiUiant ,followed by p55 antibody.The result show that not only the priming -boost stnmgy is Very effective but also the M-PG exhibite an exceuent inmunogenicity. We also have constmcted two shuttle vector plw9,pscllml which can recombine to FPV genome .The different of the two plasmid lied in different Promotors ,since strong paomotor may paly a important role in gene expression in Virat vector .But the construction of rFPV is not Over .the next work is to finish the construction.screen and purify the rFPV,transfer into cells and compare the transcription effect of the two paomotors.