携带pcDNA3s与EGFPC3质粒的重组减毒沙门氏菌的免疫性与EGFP的表达

Immunity and EGFPC3 expression of recombinant attenuated salmonella typhimurium carrying plasmid pcDNA3s and EGFPC3 in mice

目的研究HBsAg DNA重组减毒鼠伤寒沙门氏菌的免疫性与增强型绿色荧光蛋白(EGFP)基因EGFPC3在小鼠体内的表达。方法将质粒pcDNA3s与pEGFPC3分别电转化减毒鼠伤寒沙门氏菌SL8786,构建重组减毒鼠伤寒沙门氏菌SL8786/EGFPC3与SL8786/pcDNA3s。利用时间分辨荧光免疫分析技术(TRFIA)检测小鼠的血清抗体的含量,以及重组菌引起的T细胞增殖和细胞毒性T淋巴细胞(CTL)应答。用荧光显微镜观察EGFPC3表达。结果口服SL8786/p cDNA3s免疫小鼠后,可诱导产生较强的特异性CTL应答。口服免疫小鼠后第11周抗-HBs含量最高。用流式细胞术检测贴壁培养的2只小鼠的脾细胞中SL8786/EGFPC3阳性率分别为19.20%和17.36%,而SL8786/pcDNA3口服的2只小鼠脾细胞中的EGFP阳性率分别仅为1.95%和1.63%。给小鼠口服SL8786/EGFPC33周后,在小鼠肝脏、脾脏、肾脏、胸腺、十二指肠及肌肉等组织中,均有EGFPC3的表达。结论口服SL8786/pcDNA3s在小鼠体内诱导了特异性细胞和体液免疫应答。携带EGFPC3的重组鼠伤寒沙门氏菌SL8786/EGFPC3在小鼠的部分组织中产生绿色荧光表达,该重组菌的构建为减毒沙门氏菌作为活载体基因工程疫苗的研制提供了一个优良模型。

AIM： To study the immune characterization of HBsAg DNA recombinant attenuated salmonella typhimurium vaccine and the expression of enhanced green fluorescence protein gene pEGFPC3 in mice. METHODS： Plasmid pcDNA3s and pEGFPC3 was transformed into attenuated salmonella typhimurium SL8786 by electricity instrument to construct recombinant vaccine SL8786/EGFPC3 and SL8786/pcDNA3s. Mice were immunized with the recombinant vaccine and the quantity of anti-HBs antibody in the sera was measured by time resolved fluorecence immunanssay（TRFIA）,While the cytotoxocity of CTL was measured by LDH release assay. RESULTS： It produced strong specificity CTL response after taking SL8786/pcDNA3s orally to mice and anti-HBs was highest in 11 weeks. EGFPC3 expression in two mice spleen cells was detected by flow cytometry（FCM）. EGFP masculine ratio is 19. 20% and 17.36% respectively, but SL8786/pcDNA3 in two mice spleens cell EGFP masculine ratio only is 1.95% and 1.63%. The mice was taken SL8786/EGFPC3 orally after three weeks, fluorescence expression in its liver, spleen, kidney, chest gland, duodenum, muscle, etc. were observed. CONCLUSION： HBV DNA vaccine can induce specific cellular and humoral immune responses in vivo in mice by oral. Recombinant attenuated salmonella typhimurium SL8786/ EGFPC3 we constructed. It has green fluorescence expression in mice＇s partial organization, and it offers a model for research gene engineering vaccine .