摘要
目的研究塞姆利基森林病毒(Semliki forest virus,SFV)衣壳蛋白5′翻译增强区对基于该病毒复制子的HIVGagDNA疫苗抗原表达水平和免疫原性的影响。方法将SFV衣壳蛋白(capsid,C蛋白)基因的5′端102bp翻译增强区插入到SFV复制子DNA疫苗载体pCMVRep的SFV亚基因启动子下游,得到DNA疫苗载体pCMVRepC。将删除ATG的HIV1gag基因插入pCMVRepC,使gag编码区与翻译增强区融合,得到DNA疫苗质粒pCMVRepCgag。同时,构建携带未融合翻译增强序列的DNA疫苗质粒pCMVRepgag。Westernblot检测翻译增强序列对Gag表达水平的影响。用上述两种DNA疫苗分别免疫BALBc雌性小鼠,ELISA检测Gag特异的抗体反应,ELISPOT和细胞内因子染色技术检测细胞免疫应答。结果衣壳蛋白5′翻译增强区增强了Gag表达水平,对体液免疫应答没有显著影响,但显著增强了特异性细胞免疫应答水平。结论SFVC蛋白翻译增强区能显著提高SFV复制子DNA疫苗的抗原表达和抗原特异性细胞免疫反应。
Objective To investigate the effects of 5＇ translation-enhancing region of Semliki forest virus （SFV） capsid protein gene on the gene expression and immunogenicity of HIV Gag DNA vaccine based on SFV replicon DNA vector. Methods Plasmid vector pCMV-RepC was constructed by inserting the 102 bp translationenhancing region of SFV capsid gene into the downstream of subgenomic promoter of a SFV-replicon based DNA vaccine vector pCMV-Rep. DNA vaccine plasmid pCMV-RepC-gag was constructed by inserting the start codon ATC-removed gag gene into pCMV-RepC and fused to the translation-enhancing region. Another DNA vaccine plasmid pCMV-Rep-gag which expresses natural Gag antigen was also constructed by cloning the gag gene into pCMV-Rep. Expression levels of Gag antigen from the two constructs were compared by transfection of mammalian cells and Western blot analysis. Three groups of female BALB/c mice were i. m injected with pCMV-RepC-gag, pCMV-Rep-gag and pCMV-Rep, respectively. Gag-specific antibodies were detected by ELISA. Gag-specific cellular immtme responses were detected by ELISPOT and intracellular cytokine staining. Results Expression level of Gag antigen and Gag-specific cellular immune responses were significantly increased by the fusion of the translation enhancing region. However, Gag-specific antibody responses were not obviously affected. Conclusion Antigen expression and cellular immune responses induced by the HIV Gag DNA vaccine based on a SFV replicon DNA vector were significantly improved by fusing the translation-enhancing region of SFV capsid gene to the 5＇-terminus of gag gene.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2005年第8期672-676,共5页
Chinese Journal of Microbiology and Immunology
基金
国家高技术研究发展计划(863计划
2003AA219100)
美国NIH"中国综合性艾滋病研究项目"(CIPRA)
