5个HIV基因修饰可明显提高其在不同系统中的表达

目的:确定HIV-1疫苗中有效的交叉保护性细胞免疫抗原,提高各个基因在相应疫苗载体中的表达水平,为研究不同抗原在DNA载体和痘苗病毒载体中的免疫原性奠定实验基础。方法:选择HIV B′/C亚型5个以细胞免疫为主的抗原(Gag、Pol、Rev、Tat和Nef),进行基因序列优化及表达结构改造,并分别构建以质粒DNA和重组痘苗病毒为载体的两大类HIV-1疫苗。结果:优化前后5个目的基因均能够在这2种载体中有效表达;虽然采用相同的基因修饰策略,但与痘苗病毒载体相比,在DNA载体中各基因表达水平的提高均较为明显;含有抑制性序列(INS)的gag、pol基因经密码子优化后,Gag、Pol蛋白的表达均明显提高,其中Pol蛋白的提高更为明显,单独pol基因比gagpol天然结构表达水平要高,而gag基因却变化不大;对于rev、tat、nef基因而言,优化后的单独基因结构要略高于优化后的融合结构(hRTN),且二者均高于未优化的融合结构(RTN)。结论:为进一步确定HIV-1疫苗中有效的交叉保护性细胞免疫抗原、研究不同抗原在DNA载体和痘苗病毒载体中的免疫原性奠定了实验基础,为进一步研究DNA疫苗和重组痘苗病毒疫苗联合免疫提供了实验依据。

Effect of Five HIV Genes Modification on Antigen Expression in Different System

Objective: To pick out the cross protective cell mediated immunity antigens and enhance the expres sion of HIV genes in the vaccine.Methods: We selected five major cellular immune antigens of HIV-1 B′/C sub type as target genes,which are gag,pol,rev,tat and nef,to modify and optimize their gene sequences,codon bi as and express structure,and then constructed DNA vaccines or recombinant vaccinia virus(rVV) to assess the ex press level of the genes in vitro,respectively.Results: Western blot and flow cytometry(FCM) results showed that all optimized and non-optimized genes can express in DNA and rVV.Although using the same genetic modifica tion strategies,genes expressed better in DNA than rVV.As for the gag and pol,including INS sequence,had higher express level after gene optimization.The significant promote of express level was observed in pol gene,sin gle pol gene had higher express level than gagpol fusing gene,but similar result was not observed in gag.As for rev,tat and nef,optimized single gene expressed slight higher level than optimized hRTN(fusing gene by rev,tat and nef),and both optimized single rev,tat,nef gene and fusing hRTN had better express level than non-opti mized RTN.Conclusion: These data suggest that all the optimized HIV genes may be good vaccine candidate anti gens for use as a protective HIV vaccine based on the cellular mechanism.The research also provided important evidence for the immunogenicity of different antigens on the DNA and vaccinia virus.