融合表达淋巴细胞脉络丛脑膜炎病毒和卵清白蛋白CD8+T细胞表位的减毒鼠伤寒沙门氏菌的构建与鉴定

依据淋巴细胞脉络丛脑膜炎病毒(LCMV)主要保护性抗原CD8 T细胞表位VRRPQASGVYMGNLTAQ和卵清白蛋白(OVA)CD8 T细胞表位SIINFEKL,设计、合成两条编码LCMV和OVA CD8 T细胞表位的寡核苷酸片段,经退火后,克隆入绿色荧光蛋白表达质粒pYAGFP,经PCR扩增和序列测定分析,证实成功构建T细胞表位与绿色荧光蛋白融合表达的重组质粒pYAGFPL-O,将此重组质粒pYAGFPL-O转化减毒鼠伤寒沙门氏菌X4550,获得重组沙门氏菌X4550(pYAGFPL-O).用SDS-PAGE电泳测得重组菌表达的融合蛋白约为30kD.同时,荧光显微镜下观察到X4550(pYAGFPL-O)发出黄绿色荧光.这些结果表明LCMV和OVA T细胞表位已成功表达.重组菌X4550(pYAGFPL-O)的获得为研究沙门氏菌载体携带外源抗原的T细胞应答规律及调控机理打下了重要基础.

Costruction and identification of avirulent Samlmonella typhimurium expressing CD8+ T cell epitopes of lymphocytic choriomentingitis virus and ovalbumin fused in green fluorescent protein

Based on the sequence of CD8~ T cell epitopes of lymphocytic choriomeningitis vrius (LCMV) and ovalbumin (OVA), two oligo-DNA fragements encoding LCMV CD8~ T cell epitope VRRPQASGVYMGNLTAQ and OVA CD8~ T cell epitope SIINFEKL were designed , synthesized and cloned into plasmid pYAGFP at the 5'terminal of GFP gene. The recombinant plasmid was designated as pYAGFPL- O, and further verified by sequencing the insert fragement, and then pYAGFPL-O was transferred into avirulent Salmonella typhimurium X4550, the recombinant bacteria were screened and named as X4550(pYAGFPL-O). The expressed fusion protein from X4550(pYAGFPL-O) was 30 KD as determined by SDS-PAGE, and X4550(pYAGFPL-O) shows strongly green fluorescence under the fluorescent microscope. These results showed that the CD8~ T cell epitopes of LCMV and OVA were successfully expressed in recombinant Salmonella, and it would be very useful to define the mechanism of T cell responses induced by Salmonella vehicle carrying foreign antigen.