中国HIV-1 CRF01_AE流行株结构基因和调节／辅助基因DNA疫苗的构建和免疫原性研究  

lmmunogenicity of DNA vaccines encoding structural proteins and regulatory/accessory proteins derived from an HIV-1 CRF01_AE isolate circulating in China

作　　者：袁松华[1] 万延民[1] 仇超[2] 张聪优[1] 黄杨[1] 乔勇[1] 叶芮琪[1] 邱趁丽[1] 张晓燕[2] 徐建青[2] 

机构地区：[1]上海市(复旦大学附属)公共卫生临床中心,201508 [2]复旦大学生物医学研究院

出　　处：《中华微生物学和免疫学杂志》2010年第4期355-359,共5页Chinese Journal of Microbiology and Immunology

基　　金：第四十四批中国博士后科学基金二等资助（20080440571）;国家“十一五”重大专项艾滋病专项创新性疫苗研究（2008ZX10001-012）

摘　　要：目的构建表达gag—env融合基因和tat-rev-integrase（c-half）-vif-nef融合基因的DNA疫苗，并评价其免疫原性。方法按人源密码子使用频率对AE2f株的gag、env、tat、rev、integrase、vif和nef基因序列进行优化，构建真核表达质粒。用Westernblot法验证体外表达情况；用ELISPOT法检测小鼠的细胞免疫反应。结果限制性酶切及DNA测序结果表明两个融合基因质粒构建正确，可以表达相应的融合蛋白。ELISPOT结果显示，Gag—Env特异性的T细胞反应强度为（3010±566）SFC／10^6脾细胞；Tat-Rev—Integrase（C．half）-Vif-Nef融合蛋白特异性的T细胞反应为（948±737）SFC／10^6脾细胞，均显著高于空载体组。结论构建的表达HIV-1CRF01_AE流行株gag—env融合基因和tat—rev-integrase（c-half）-vif-nef融合基因的DNA疫苗可以正确表达所编码的融合蛋白并有效地激活机体的T细胞免疫反应。Objective To construct two DNA vaccines encoding Gag-Env fusion protein and Tat-Rev-Integrase（C-half） -Vif-Nef fusion protein derived from the first HIV-1 CRF01_AE isolate（AE2f） in China and to evaluate the immunogenicity in mice. Methods Two DNA vaccines were constructed by inserting the codon optimized and synthesized gag-env fusion gene and tat-rev-integrase（c-half） -vif-nef fusion gene derived from AE2f into mammalian expression vector pDRVISV1.0, the generated DNA vaccines were designated as pSVAE/GE and pSVAE/TRIVN, respectively, and their in vitro expression were determined by Western blot with transfected 293T cells. Mice were i. m. immunized with either pDRVI1.0 as mock control, pSVAE/GE or pSVAE/TRIVN for 4 times at two-week interval. Two weeks following the final immunization, cellular responses to pool of HIV-1 Env, Gag, Tat, Rev, Intergrase, Vif and Nef peptides were evaluated by ELISPOT assay. Results The construction of DNA vaccine pSVAE/GE and pSVAE/TRIVN was validated by restriction enzyme digestion and bidirectional sequencing. Western blot showed a specific band at molecular mass 220 × 10^3 in lane of pSVAE/GE transfected 293T cell and a specific band at 95 × 10^3 in the lane of pSVAE/TRIVN. Both DNA vaccines mounted significant specific T cell responses with （3010±566） SFC/10^6 splenocytes for DNA vaccine pSVAE/GE and （948 ±737） SFC/10^6 splenocytes for DNA vaccine pSVAE/TRIVN, whereas the mock control of pDRVISV1.0 only raised marginal T cell responses. Conclusion Both pSVAE/GE and pSVAE/TRIVN were capable of expressing the inserted fusion immunogen genes and able to elicit vigorous cellular immune responses, therefore, these DNA vaccines are highly immunogenic.

关 键 词：HIV-1 艾滋病疫苗 结构基因 调节基因 

分 类 号：R392.1[医药卫生—免疫学]