表达绿色荧光蛋白的单核细胞增生李斯特菌重组菌株的构建及其生物学特性  

Construction of the recombinant strains of Listeria monocytogenes expressing green fluorescent protein and characterization of their biological properties

作　　者：殷月兰[1] 焦新安[1] 杨芸[1] 张晨菊[1] 周海霞[1] 潘志明[1] 黄金林[1] 

机构地区：[1]扬州大学,江苏省人兽共患病学重点实验室,扬州225009

出　　处：《中国人兽共患病学报》2009年第11期1037-1040,共4页Chinese Journal of Zoonoses

基　　金：973课题(2006CB504404);国家自然科学基金(30425031);扬州大学大学生学术科技创新基金项目

摘　　要：目的为了对单核细胞增生李斯特菌运送外源抗原所诱导免疫应答特性进行评价,开展了以原核方式运送模式蛋白GFP的研究。方法利用SOEing PCR的方法把LLO的启动子与GFP融合在一起,通过同源重组的方式整合到yzuLM1-2actA和plcB基因片段之后,在LLO启动子的作用下实现GFP的表达。结果PCR扩增证实目的基因gfp融合到李斯特菌基基因组中,重组菌对小鼠的LD50为4.31×108,毒力比yzuLM1-2显著降低。以重组菌株免疫小鼠后获得的血清进行Western blot显示在27kD处出现特异印迹带;ELISA测定结果显示能诱导小鼠产生较高的抗GFP的抗体水平。结论研究结果表明减毒LM所运送的GFP能诱导小鼠产生较强的免疫应答的特性,这为减毒株yzuLM1-2运送外源保护性抗原的研究奠定了基础,也为开展减毒重组菌与抗原递呈细胞之间的相互作用及其诱导的免疫应答分析提供了必要条件。To evaluate the immune responses induced by transmission of the exogenous antigens through Listeria monocytogenes in the study of prokaryotic transmission model of green fluorescent protein（GEP）,the promoter of listeria lysine O（LLO） and the GFP gene gfp were fused together by using the SOEing PCR and then integrated to yzuLM1-2 actA and plcB gene fragments through homologous recombination.In this way,GFP was expressed through the function of up-stream LLO promoter.It was verified by PCR amplification that gfp gene could be successfully integrated to listerial genome.The result in animal experiment showed that the LD50 of the recombinant bacteria in mice was 4.31×108,that was significantly reduced to 10 folds in comparison with that of the attenuated strain yzuLM1-2.The sera of mice immunized with recombinant bacteria showed specific band at 27 kDa in Western blot assay and could elicit production of high level of anti-GFP antibodies as demonstrated by ELISA assay.It is evident that the GFP transmitted by the attenuated L.monocytogeneses can induce strong immunogenicity in mice,thus constituting a solid basis to elucidate the molecular mechanism of pathogenesis and immunity triggered by L.moncytogenes.

关 键 词：单核细胞增生李斯特菌 绿色荧光蛋白 重组菌株 生物学特性 

分 类 号：R378.99[医药卫生—病原生物学]