中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2010年
4期
355-359
,共5页
袁松华%万延民%仇超%张聪优%黄杨%乔勇%叶芮琪%邱趁丽%张晓燕%徐建青
袁鬆華%萬延民%仇超%張聰優%黃楊%喬勇%葉芮琪%邱趁麗%張曉燕%徐建青
원송화%만연민%구초%장총우%황양%교용%협예기%구진려%장효연%서건청
HIV-1%艾滋病疫苗%结构基因%调节基因
HIV-1%艾滋病疫苗%結構基因%調節基因
HIV-1%애자병역묘%결구기인%조절기인
HIV-1%AIDS vaccine%Structural gene%Regulatory gene
目的 构建表达gag-env融合基因和tat-rev-integrase(c-holf)-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Western blot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-1 CRF01_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.
目的 構建錶達gag-env融閤基因和tat-rev-integrase(c-holf)-vif-nef融閤基因的DNA疫苗,併評價其免疫原性.方法 按人源密碼子使用頻率對AE2f株的gag、env、tat、rev、integrase、vif和nef基因序列進行優化,構建真覈錶達質粒.用Western blot法驗證體外錶達情況;用ELISPOT法檢測小鼠的細胞免疫反應.結果 限製性酶切及DNA測序結果錶明兩箇融閤基因質粒構建正確,可以錶達相應的融閤蛋白.ELISPOT結果顯示,Gag-Env特異性的T細胞反應彊度為(3010±566)SFC/10~6脾細胞;Tat-Rev-Integrase(C-half)-Vif-Nef融閤蛋白特異性的T細胞反應為(948±737)SFC/10~6脾細胞,均顯著高于空載體組.結論 構建的錶達HIV-1 CRF01_AE流行株gag-env融閤基因和tat-rev-integrase(c-half)-vif-nef融閤基因的DNA疫苗可以正確錶達所編碼的融閤蛋白併有效地激活機體的T細胞免疫反應.
목적 구건표체gag-env융합기인화tat-rev-integrase(c-holf)-vif-nef융합기인적DNA역묘,병평개기면역원성.방법 안인원밀마자사용빈솔대AE2f주적gag、env、tat、rev、integrase、vif화nef기인서렬진행우화,구건진핵표체질립.용Western blot법험증체외표체정황;용ELISPOT법검측소서적세포면역반응.결과 한제성매절급DNA측서결과표명량개융합기인질립구건정학,가이표체상응적융합단백.ELISPOT결과현시,Gag-Env특이성적T세포반응강도위(3010±566)SFC/10~6비세포;Tat-Rev-Integrase(C-half)-Vif-Nef융합단백특이성적T세포반응위(948±737)SFC/10~6비세포,균현저고우공재체조.결론 구건적표체HIV-1 CRF01_AE류행주gag-env융합기인화tat-rev-integrase(c-half)-vif-nef융합기인적DNA역묘가이정학표체소편마적융합단백병유효지격활궤체적T세포면역반응.
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 Chi-na 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 de-rived from AE2f into mammalian expression vector pDRVISV1. 0, the generated DNA vaccines were desig-nated 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 pSYAE/TRIVN for 4 times at two-week interval. Two weeks following the final im-munization, 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 transfeeted 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 pSV AE/GE and (948 ± 737) SFC/10~6 spleno-cytes 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 vac-cines are highly immunogenic.
