CAJ | 학술논문

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.
목적 구건표체함유밀마자우화적HIV-1 CRF01_AE아형env기인적DNA역묘,위다재체애자병치료성역묘적응용제공후선역묘.방법 대HIV-1감염자혈액양본진행형별분석,분형득도적AE아형표본채용아형특이성인물극륭env기인,통과서렬비대획득기공유서렬,대해서렬안조포유동물밀마자사용적편기성진행우화,장인공합성적우화기인극륭지pVR재체,구건DNA역묘.통과Western Blot방법비교우화전후env기인적표체.결과 성공획득32조구유완정적개방성열독광적env극륭,형별분석학인균위CRF01_AE아형.성공대기공유서렬진행료우화、합성병구건료DNA역묘pVR-mod.AE env,해역묘여야생형적env기인(wt.AE env)상비가이고수평표체env기인,차불의뢰Rev적존재.결론 대HIV-1중국류행주CRF01_AE env기인적우화개조급중조DNA역묘적구건시성공적.
Objective 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.