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.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
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）
