猪霍乱沙门氏菌递送的双启动子表达载体的构建

Construction of a dual-promoter expression plasmid delivered by Salmonella choleraesuis C500

摘要:

猪霍乱沙门氏菌C500株不仅可以作为预防猪沙门氏菌病的活疫苗, 还可作为运送其他DNA疫苗的优良载体,并通过粘膜免疫诱导产生针对特定抗原的各种免疫应答。为增强其携带的DNA疫苗的免疫效力, 本研究以真核表达载体pEGFP-C1为基础, 将其真核启动子CMVIE与原核启动子Ptrc串联, 并在其多克隆位点MCS下游引入rrnbT1T2转录终止序列, 构建了真、原核双启动子表达载体pEGFPPtrcR。用1×TSS法将其转化C500, 得到工程菌C500/pEGFPPtrcR, 通过SDS-PAGE和Western blotting鉴定了报告基因EGFP的原核表达, 该菌在荧光显微镜下能发出强烈绿色荧光, 被证明在体外至少能稳定遗传20代; 采用脂质体介导法将pEGFPPtrcR转染Vero细胞, EGFP在胞核和胞浆内表达, 24 h后观察可到明显绿色荧光。结果表明, 双启动子表达载体pEGFPPtrcR构建成功, 预示其携带的外源基因既可在C500中表达, 又可在体细胞中表达, 为研制以C500为载体的新型DNA疫苗的发展开辟了一个新的途径。

Abstract:

Salmonella choleraesuis C500 strain is an attenuated vaccine preventing piglet from paratyphoid and can also be used as a live vector of other DNA vaccines. Through mucosal immunization, immune response to specific antigens carried by it can be induced. To enhance the immune efficiency of DNA vaccine it carried, promoter Ptrc was inserted into the down stream of the CMV immediate early promoter of eukaryotic expression plasmid pEGFP-C1. Then transcription terminator rrnbT1T2 was inserted into down stream of the multiple clone sites of pEGFP-C1, and the dual-promoter expression vector pEGFPPtrcR was constructed. Using 1×TSS method, the recombinant plasmid was transformed into C500, C500/pEGFPPtrcR was obtained. SDS-PAGE and Western blotting was used to detect the expression of report gene EGFP. Strong green fluorescence can be observed under fluorescent microscope. The stable passages of this recombinant bacterium were at least 20 generations in vitro. The plasmid pEGFPPtrcR was transfected into vero cell using liposome. After 24 h, green fluorescent was observed, showing the expression of EGFP in nuclei and endochylema. The result manifested that the construction of dual-promoter expression vector pEGFPPtrcR was successful. It also indicated that the foreign gene was expressed in salmonella strain C500 and somatocytes, resulting in increased antigen expression. This research provided a foundation for the research of new DNA vaccines using salmonella C500 as carrier.