携带 EGFPC3 质粒的重组减毒鼠伤寒沙门氏菌在小鼠体内的表达

目的 构建重组减毒鼠伤寒沙门氏菌X8786/pEGFP-C3,检测增强型绿色荧光蛋白C3(EGFP-C3)在小鼠体内荧光表达.方法 将质粒pEGFP-C3电转化到减毒鼠伤寒沙门菌SL8786,构建重组减毒鼠伤寒沙门菌X8786(pEGFP-C3),分别灌胃饲服昆明种小鼠,流式细胞仪检测脾脏细胞荧光表达,利用荧光显微镜检测小鼠组织荧光表达.结果 在体外稳定试验中,重组菌经LB固体及液体培养基连续传15代后,单个菌落和菌液均呈绿色;流式细胞仪结果证实,体外情况下,减毒鼠伤寒沙门菌可以将pEGFP-C3转入小鼠腹腔灌洗巨噬细胞,并在其中表达.在体内稳定试验中,经昆明种小鼠连续传代10次,从肝脏、脾脏中分离的细菌经LB固体培养仍为绿色,证明构建的重组减毒鼠伤寒沙门氏菌是稳定的.用免疫剂量的重组细菌接种昆明种小鼠后,观察1个月未见有异常现象,同时剖检后也未见脏器有眼观病变.免疫一定时间后,在小鼠肝脏、脾脏、肾脏、胸腺、十二指肠、肌肉等组织有荧光表达.结论 表达EGFP-C3的重组鼠伤寒沙门氏菌的成功构建为减毒沙门氏菌作为活载体的基因工程疫苗研制提供一个优良模型.

Expression of recombinant attenuated salmonella typhimurium carrying enhancement green fluorescence protein in mice

Objective To construct recombinant attenuated salmonella typhimurium X8786(pEGFP-C3) and detect the expression of enhanced green fluorescent protein gene (EGFP-C3) in mice. Methods In order to construct recombinant attenuated salmonella typhimurium X8786/pEGFP-C3, plasmid pEGFPC3 was translated to attenuated salmonella typhimurium SL8786 by electricity instrument. The recombinant X8786/pEGFP-C3 was fed to six weeks old mice. The EGFP-C3 expression and the organization fluorescent expression in mice were detected by flow cytometry and fluorescent microscopy, respectively. Results The recombinant strains cultured in solid and liquid LB medium through 15 generations still expressed GFP correctly and illuminated green fluorescent. The EGFP-C3 expressed in macrophage cell from mouse peritoneal lavage was detected by flow cytometry, so did the recombinant strains inoculated in mice and then separated from liver and spleen. The recombinant attenuated salmonella typhimurium had considerable stability both in vitro and in vivo. Visible pathological changes of internal organs were not observed in mice after inoculation with immune dosage of recombinant bacteria for one month. The fluorescence expression was found in mouse liver, spleen, kidney, chest gland, duodenum, muscle, etc. after immunization for a certain time. Conclusion The recombinant attenuated salmonella typhimurium expressing EGFP-C3 is constructed, which offers a fine model for the research of genetic engineering vaccine of the living carrier.