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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 study the effect of in vivo electroporation delivery on reporter gene expression levels and immune responses induced by HIV Gag DNA vaccine in a mouse model. Methods Luciferase expression plasmid p1.0-luc was constructed and injected directly by intramuscular injection, then electroporated with double-needle electrode after intramuscular injection, and then electroporated with forceps electrode after intramuscular injection Mice, 72h after injection of the muscle to determine the expression of luciferase. At the same time, a DNA vaccine plasmid p1.0-gag carrying the gag gene of codon optimized HIV-1B ’/ C CN53 strain was constructed and immunized BALB / c by the above three different methods at two dose levels of 10μg and 100μg Female mice were tested for Gag-specific antibody response by ELISA, and cellular immune responses were detected by ELISPOT and jntracellular cytokine staining (ICS). Results By in vivo electroporation, the expression of luciferase in mouse muscle significantly increased by up to 66 times. Gag DNA vaccine immunization results showed that electroporation could significantly increase Gag-specific humoral immune response, the use of double-needle electrode effect was significantly better than the clamp electrode, the former induced antibody titers than the non-use of electroporation group increased up 28 times. However, in vivo electroporation did not have a significant effect on Gag-specific cellular immune responses. Conclusion In vivo electroporation (especially using the double-needle electrode) can greatly enhance the expression level of the reporter gene in vivo and the antigen-specific humoral immune response induced by the DNA vaccine.