含pIRES2-EGFP-4-1BBL质粒减毒沙门氏菌株的构建与鉴定  

Construction and indentification of the recombinant vaccine of Attenuated Salmonella typhimurium containing pIRES2-EGFP-4-1BBL vector

作　　者：叶建新[1] 仉元亭[1] 陈卫昌[1] 张学光[2] 任大明[3] 

机构地区：[1]苏州大学第一医院消化科江苏省临床免疫重点实验室,苏州215006 [2]苏州大学生物技术研究所,苏州215007 [3]复旦大学遗传学研究所遗传工程国家重点实验室,上海200433

出　　处：《中国免疫学杂志》2009年第4期336-339,345,共5页Chinese Journal of Immunology

基　　金：江苏省临床免疫重点实验室基金;江苏省自然科学基金(BK20088171);江苏省研究生科研创新基金(2008年)资助

摘　　要：目的:构建含pIRES2-EGFP-4-1BBL质粒的SL3261(减毒鼠伤寒沙门氏菌)疫苗菌。方法:将质粒pIRES2-EGFP-4-1BBL转入LB5000(鼠伤寒沙门氏菌)进行甲基化,利用修饰后重组质粒转化终宿主菌SL3261,抗生素筛选后挑取单个菌落扩增进行抽提质粒和PCR鉴定,SL3261疫苗菌与HepG2细胞体外共培养,荧光显微镜和RT-PCR分别检测GFP(绿荧光蛋白)的表达和4-1BBL基因的转录。结果:导入pIRES2-EGFP-4-1BBL质粒的SL3261经抽提质粒电泳后获得了同原转化质粒相同大小的目的条带,质粒PCR获得了约930bp的条带,200个HepG2细胞感染SL3261为201±46CFU、SL3261-pIRES2-EGFP为163±37,而SL3261-pIRES2-EGFP-4-1BBL为158±32,含质粒SL3261在体外感染HepG2细胞的能力同原细菌基本相同(P>0.05)。感染含质粒SL3261的HepG2细胞观察到GFP表达,感染含重组质粒SL3261的HepG2细胞经RT-PCR检测到约930bp的目的条带。结论:成功构建了能携带大鼠4-1BBL基因真核表达质粒的SL3261疫苗菌。Objective：To develop a strain of vaeeine in Attenuated Salmonella typhimurium SL3261 harboring recombinant expressing vector of rat 4-1BBL gene. Methods： pIRES2- EGFP-4-1BBL vector was converted to LB5000（ Salmonella typhimurium） for methylation and then the modified eukaryotic vector was electrotransfered to final host SL3261, screened single colonies by antibiotics was tested through plasmid extraction and PCR. Meanwhile the reconstructed SL3261 or its predecessor was cocultured with HepG2 cells to detect their invasion. Fluorescence microscope observasion and RT-PCR was exploited to ohserve GFP and 4-1BBL mRNA in the host cells. Results：The similar size of originated plasmids was extracted from strains SL3261 imported by the pIRES2-EGFP-4-1BBL plasmids, which was amplified as a 930 bp fragment by PCR. The capacity of SL3261 to invade HepG2 cells was 201 ± 46 CFU/200 HepG2 cells, whereas SL3261-pIRES2-EGFP was 163 ± 37 CFU/ 200 HepG2 cells and SL3261-pIRES2-EGFP-4-1BBL was 158 ±32 CFU/200 HepG2 cells（ P 〉 0. 05）. The transfected HepG2 cells by infection of SL3261 containing the vector were shovel with GFP and the 930 bp target expression by RT-PCR. Conclusion： Attenuated Salmonella typhimurium SL3261 containing recombined eukaryotic expressing plasmid pIRES2-EGFP-4-1BBL is successfully constructed which can deliver recombinant plasmid into HepG2 cells.

关 键 词：4-1BBL基因 减毒沙门氏菌 DNA疫苗 

分 类 号：R392.11[医药卫生—免疫学]