摘要目的 构建表达中国主要流行亚型HIV-1 tat-rev-integrase(c-half)-vif-nef(TRIVN)融合基因的DNA疫苗,并比较其免疫原性.方法 按人源密码子使用频率对HIV-1 CN54(B'/C重组亚型)与RL42(B'亚型)的tat、rev、integrase(C端144个氨基酸)、vif和nef基因序列进行优化,构建DNA疫苗.通过Western blot测定上述DNA疫苗与HIV-1 AE2f株来源的tat-rev-integrase(c-half)-vifnef融合基因DNA疫苗的体外表达效率;利用小鼠模型比较3个DNA疫苗单独免疫与混合免疫的免疫原性特征.结果 限制性酶切及DNA测序结果表明两个融合基因重组质粒构建正确;Western blot 检测结果显示:3个DNA疫苗的体外表达效率基本相当.小鼠免疫后ELISPOT检测结果显示:在总T细胞反应强度方面AE2f-TRIVN最强[(948.0±330.0)SFCs/106脾细胞],次之为混合DNA免疫组(500.0±155.0 SFCs/106脾细胞),再者为RL42-TRIVN[(195.1±44.0)SFCs/106脾细胞],CN54-TRIVN最弱[(89.5±17.0)SFCs/106脾细胞].T细胞反应分布情况显示:3个DNA疫苗单独免疫时,T细胞反应主要集中在Integrase和Vif蛋白上;而混合免疫可以部分改善针对Nef蛋白的免疫识别.结论 3个亚型TRIVN DNA疫苗中以AE2f-TRIVN的免疫原性最强;DNA疫苗混合免疫倾向于促进特异性T细胞反应在TRIVN融合抗原上的均匀分布.

abstracts0bjective To determine the immunogenicities of DNA vaccines expressing tat-rev-integrase(c-half)-vif-neffusion genes(TRIVN) derived from prevalent B', B'/C and AE recombinant subtypes of HIV-1 in China. Methods Two DNA vaccines were constructed by inserting the codon optimized tat-revintegrase(c-half)-vif-nef fusion genes derived from B' and B'/C subtype of HIV-1 into mammalian expression vector pSVI. 0. DNA vaccine containing tat-rev-integrase (c-half)-vif-nef fusion gene derived from HIV-1AE2f has been constructed previously. In vitro expression efficiencies of three DNA vaccines were determined by Western blot and their immunogenicities were compared by immunizing female BALB/c mice. IFN-γ ELISPOT assay was used to read out the specific T cell immunity. Results The constructed DNA vaccines were validated by restriction enzyme digestion and DNA sequencing. Western blot assay showed three constructed DNA vaccines could be expressed at a comparable level in vitro. After vaccination, AE-TRIVN mounted T cell immune responses at (948.0 ± 330.0) SFCs/106 splenocytes, followed by the mixed DNA vaccine[ (500.0 ± 155.0) SFCs/106 splenocytes ], RL-TRIVN r[ ( 195. 1 ± 44.0) SFCs/106 splenocytes ]and CN-TRIVN [ (89.5 ± 17.0) SFCs/106 splenocytes]. Interestingly, we observed that single DNA vaccination induced specific T cell responses predominantly targeting Integrase (C-half) and Vif, whereas the mixed DNA could significantly improve T cell responses against Nef. Conclusion AE-TRIVN was the most immunogenic among the three DNA vaccines and the mixed DNA vaccination could change the immunogenic hierarchy of T cell epitopes across the fusion genes vaccine.