减毒鼠伤寒沙门菌作基因递呈载体的体外试验

目的　体外情况下验证鼠伤寒沙门菌作为基因递呈载体 ,将基因转染入真核细胞的能力 .方法　构建增强绿色荧光蛋白 (EGFP)真核表达载体 pc DNA3.1(+) / EGFP,将重组 pc DNA3.1(+) / EGFP质粒和 pc DNA3.1(+)空载体用电穿孔法分别转入减毒鼠伤寒沙门菌 .分别用 2种重组细菌体外情况下感染小鼠腹腔灌洗巨噬细胞 ,培养 4 8h后 ,流式细胞仪检测两组小鼠巨噬细胞荧光强度 ,验证鼠伤寒沙门菌作为基因递呈载体的能力 .结果　成功构建了 pc DNA3.1(+) /EGFP重组鼠伤寒沙门菌 ;感染细胞培养 4 8h后 ,流式细胞仪检测表明 pc DNA3.1(+) / EGFP重组减毒鼠伤寒沙门菌感染的小鼠巨噬细胞荧光细胞百分比为 4 0 .6 % ,荧光强度为0 .92 7,均明显高于对照组 (分别为 3.8% ,0 .345 ,P<0 .0 1) .结论　减毒鼠伤寒沙门菌是一种有效的基因递呈工具 ,可将外源基因转入真核细胞并在其中表达 ,为进一步应用其研制口服 DNA疫苗奠定了基础 .

Attenuated Salmonella typhimurium as DNA delivery system in vitro

AIM To test the ability of Salmonella typhimurium to transfer gene to eukaryotic cells in vitro . METHODS The eukaryotic expression vector of enhanced green fluorescence protein (EGFP), pcDNA3.1(+)/EGFP, was constructed and then transduced into attenuated Salmonella typhimurium by electroperforation. As negative control, the empty vector pcDNA3.1(+) was also transduced into the same strain of Salmonella typhimurium . The two kinds of Salmonella transfectants were then used to infect murine peritoneal macrophages in vitro respectively. After 48 h of incubation, the cells were examined by flow cytometry to determine the expression of EGFP by the pcDNA3.1(+)/EGFP recombinant Salmonella infected cells. RESULTS The pcDNA3.1(+)/EGFP plasmid was successfully transduced into S. Typhimurium. by FCM examination. It was shown that in the mcrophage cells infected by Salmonella typhimurium harboring the pcDNA3.1(+)/EGFP plasmid, the percentage of fluorescent cells was 40.6%, and fluorescence intensity was 0.927, which were significantly higher than those of the control. (which were 3.8% and 0.345 respectively, P < 0.01). Our findings suggested that EGFP was expressed in the infected murine peritoneal macrophage cells. CONCLUSION Attenuated Salmonella typhimurium can transfer exogenous gene to the eukaryotic cells as a carrier for oral DNA vaccines.