体内电穿孔对报告基因表达和HIV Gag DNA疫苗诱导的免疫反应的影响  

Effects of in vivo electroporation on reporter gene expression and immune responses induced by DNA vaccination

作　　者：李鼎锋[1] 张玉伟[1] 李海山[1] 范文玲[1] 孙茂盛[2] 刘勇[1] 邵一鸣 

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心,北京100050 [2]中国医学科学院中国协和医科大学医学生物学研究所

出　　处：《中华微生物学和免疫学杂志》2006年第11期1038-1041,共4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)

摘　　要：目的研究体内电穿孔递送途径在小鼠模型中对于报告基因表达水平及HIV Gag DNA疫苗所诱导的免疫反应的影响。方法构建荧光素酶(luciferase)表达质粒p1．0-luc,将其通过直接肌肉注射、肌注后以双针电极进行电穿孔、肌注后以钳式电极进行电穿孔等3种不同方法注射小鼠,72h后取注射部位的肌肉测定luciferase的表达情况。同时构建携带密码子优化的HIV-1B′/C重组亚型CN54株gag基因的DNA疫苗质粒p1．0-gag,在10μg、100μg两个剂量水平上通过以上3种不同的方法免疫BALB/c雌性小鼠,ELISA方法检测Gag特异的抗体反应,ELISPOT方法和细胞内因子染色(jntracellular cytokine staining,ICS)技术检测细胞免疫应答。结果通过体内电穿孔可以使luciferase在小鼠肌肉中的表达水平显著提高,最大提高幅度达到66倍。Gag DNA疫苗免疫结果显示,电穿孔可以显著提高Gag特异的体液免疫应答,其中使用双针电极的效果要显著好于钳式电极,前者所诱导的抗体滴度比不使用电穿孔组提高可达28倍。但体内电穿孔对于Gag特异的细胞免疫应答并没有显著影响。结论体内电穿孔(尤其是使用双针电极)可以大幅度提高报告基因在体内的表达水平和DNA疫苗诱导的抗原特异性体液免疫应答。Objective To investigate the effects of in vivo electroporation on reporter gene expression and immune responses induced by DNA vaccination. Methods Luciferase reporter plasmid pl. 0-1ue was construeted and administered intramuscularly to BALB/e mice through injection without electroporation, injection plus 2-needle-electrobe electroporation or injection plus tweezertrede-electrobe electroporation. Luciferase expression level in murine muscle was detected 72 hours after injection. DNA vaccine plasmid p1.0-gag carrying codon-optimized gag gene of CN54 strain（HIV-1 B＇/C recombinant） was administered to mice at dosages of 10 μg or 100μg through the three approaches mentioned above. Mice were immunized at weeks 0, 2 and 4. Gag-specific immune responses were detected at week 6. Gag-specific antibodies were detected by ELISA. Gag-specific cellular immune responses were determined by IFN-γ ELISPOT and intracellular cytokine staining（IFN-7）. Results htciferase expression level in routine muscle was significantly increased（66 folds） by in vivo electrotransfer. Results of DNA vaccination revealed that Gag-specific humoral immune responses were significantly enhanced（28 folds） by electroporation after injection. 2-Needle electrobe was better than tweezertrede electrobe in terms of facilitating both gene expression and immunity. However, neither electroporation methods showed significant effect on cellular immune responses induced by DNA vaccination. Conclusion Reporter gone expression and humoral immune responses induced by DNA vaccination were greatly improved by in vivo electroporation after intramuscularly injection of plasmid.

关 键 词：体内电穿孔 DNA疫苗 基因表达 

分 类 号：R392[医药卫生—免疫学]