摘要目的 构建表达含有密码子优化的HIV-1 CRF01_AE亚型env基因的DNA疫苗,为多载体艾滋病治疗性疫苗的应用提供候选疫苗.方法 对HIV-1感染者血液样本进行型别分析,分型得到的AE亚型标本采用亚型特异性引物克隆env基因,通过序列比对获得其共有序列,对该序列按照哺乳动物密码子使用的偏嗜性进行优化,将人工合成的优化基因克隆至pVR载体,构建DNA疫苗.通过Western Blot方法比较优化前后env基因的表达.结果 成功获得32条具有完整的开放性阅读框的env克隆,型别分析确认均为CRF01_AE亚型.成功对其共有序列进行了优化、合成并构建了DNA疫苗pVR-mod.AE env,该疫苗与野生型的env基因(wt.AE env)相比可以高水平表达env基因,且不依赖Rev的存在.结论 对HIV-1中国流行株CRF01_AE env基因的优化改造及重组DNA疫苗的构建是成功的.

abstractsObjective To construct a DNA vaccine containing codon-modified HIV-1 consensus env gene which will be a candidate vaccine in multiple vector-based therapeutic AIDS vaccine strategy.Methods HIV-1 CRF01_AE env genes were amplified using subtype-specific primmer sets and cloned to pcDNA 3.1.The consensus sequence was acquired by alignment of all the env sequences with software.Then codons of the consensus env sequence were modified according to mammalian codon usage.The modified env gene mod.AE env was cloned into pVR vector to get DNA vaccine pVR-mod.AE env.The expression level of wild type and codon-modified env gene was analyzed by Western Blot assay.Results Phylogenetic analysis of the full-length env nucleotide sequences confirmed that all 32 isolates were grouped within CRF01_AE.The DNA vaccine expressing the codon-modified consensus env gene derived from these 32 sequences was constructed successfully.The codon modification increased the expression level of Env protein significantly.Conclusion The codon modification of CRF01_AE env gene and construction of DNA vaccine expressing this gene was successful.