HIV-1 C亚型密码子优化gp120基因重组腺病毒载体的构建及表达

目的构建含有HIV-1C亚型gp120基因重组腺病毒载体，并在293细胞中表达gp120蛋白。方法PCR扩增，获得HIV-1C亚型gp120片段，定向克隆人腺病毒转移载体pTrack-CMV，线性化后转化至含有腺病毒骨架载体pAd-easy-1的大肠埃希菌BJ5183，获得重组子prAd—gp120，PacI酶切纯化后转染293细胞，包装成复制缺陷型重组腺病毒vAd—gp120。结果经PCR、酶切及DNA测序，插入片段大小、方向正确，获得了具有感染力的vAd—gp120重组腺病毒；通过Western印迹检测，重组腺病毒在293细胞中表达出分子量为120kD的蛋白。结论成功构建了含有HIV-1C亚型gp120基因重组腺病毒载体，并获得该基因的表达。

Construction of Replication-deficient Recombinant Adenoviral Vector Containing Codon Usage-optimized gp120 Gene of HIV-1 Subtype C and its Expression

Objective To construct replication-deficient recombinant adenoviruses containing HIV-1 subtype C gp120 gene and express gpl20 in 293 cells. Methods HIV-1 gpl20 gene fragment was obtained by PCR and cloned into the shuttle vector pTrack-CMV, and then transformed into E. coli BJ5183 containing adenoviral backbone pAd-easy-1 following linearization at a Pme I site. The resultant recombinant plasmid, named prAd-gp120, was digested with Pac I and then transfected into packaging cell line 293 cells by use of Lipofectamin 2000, leading to production of infective replication-deficient recombinant adenoviruses, vAd-gp120. Results Restriction enzyme digestion and sequencing analysis confirmed correct insertion of HIV-1 subtype C gp120 gene in pTrack-CMV vector. Expression of gpl20 in 293 cells was detected by Western Blotting assay. Conclusion The replication-deficient recombinant adenoviral vector containing gpl20 was correctly constructed. The obtained vAd-gp120 was able to express gp120 protein in 293 cells sufficiently.