共表达EGFP和UreB活载体疫苗的体内外稳定性研究  

ESTABLISHMENT OF MYCOBACTERIUM SMEGMATIS VACCINE CO-EXPRESSING HELICOBACTER PYLORI UreB AND GREEN FLUORESCENT PROTEIN

作　　者：向廷秀[1] 谯敏[2] 姜政[2] 陶小红[2] 黄爱龙[3] 王丕龙[2] 

机构地区：[1]重庆医科大学附属第一医院二病区实验室,重庆400016 [2]重庆医科大学附属第一医院消化内科,重庆400016 [3]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《西南农业大学学报（自然科学版）》2006年第6期1011-1015,共5页Journal of Southwest Agricultural University

基　　金：2004年国家自然科学基金资助项目(30371318)

摘　　要：建立共表达人幽门螺杆菌UreB和绿色荧光蛋白的重组耻垢分枝杆菌疫苗株,为进一步应用重组耻垢分枝杆菌疫苗防治幽门螺杆菌感染打下基础。利用聚合酶链反应(Polym erase chain reaction,PCR)扩增的幽门螺杆菌的尿素酶编码基因全长片段,克隆入大肠杆菌-分枝杆菌穿梭质粒pLA 73,构建pLA 73-UreB载体,将pLA 73-UreB和pEGFP共转化耻垢分枝杆菌,经卡那霉素和氨苄青霉素双筛选后,抽提质粒用PCR法鉴定,W esternb lot检测UreB的表达及活性。液体培养菌液在荧光显微镜下发出明亮的绿色荧光。从幽门螺杆菌基因组中扩增出约1 710 bp的基因片段,所构建重组体阳性克隆经酶切和PCR鉴定与预期结果一致,测序结果确证了插入片段的正确性,W estern b lot检测说明幽门螺杆菌的UreB编码基因在分枝杆菌中获得了表达并具有良好的免疫原性。成功构建了共表达EGFP和幽门螺杆菌UreB基因的耻垢分枝杆菌疫苗,为其在幽门螺杆菌的防治中的应用奠定了研究基础。To construct a recombinant Myeobacterium smegmatis me 2155 for the coexpression of th.licobacter pylori （Hp） UreB and green fluorescent protein. A recombinant Escherichia coli- Helicobacteria shuttle plasmid was constructed by gene engineering technique, identified by digestion with restriction endonuclease and PCR. The shuttle plasmid and pEGFP were co- transh）rmed into me 2155 by electric perforation. Tile expressed product was detected under a fluorescent microscope. Agarose gel electrophoresls showed that the gene was inserted into the fragment amplified by PCR. Liquid culture showed bright green fluorescence in the light of a long - wave ultraviolet ray. Recombinant ptasmid and M.smegmatis vaccine were successfully established, which lays the foundation for the research of the ap- plication of this vaccine in H. pylori immunotherapy.

关 键 词：绿色荧光蛋白 幽门螺杆菌 疫苗 耻垢分枝杆菌 

分 类 号：R735.2[医药卫生—肿瘤]