title: [Construction of a dual-promoter expression plasmid delivered by Salmonella choleraesuis C500].
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 human cytomegalovirus (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 1xTSS method, we transformed the recombinant plasmid into C500, and obtained C500/pEGFPPtrcR. We used SDS-PAGE and Western blotting to detect the expression of report gene EGFP. Strong green fluorescence was observed under fluorescent microscope. The stable passages of this recombinant bacterium were at least 20 generations in vitro. Using liposome we transfected plasmid pEGFPPtrcR into Vero cell. After 24 h, green fluorescent was observed, showing the expression of EGFP in nuclei and endochylema. The construction of dual-promoter expression vector pEGFPPtrcR was successful. The foreign gene was expressed in Salmonella strain C500 and somatocytes, resulting in increased antigen expression. This research provides a foundation for the research of new DNA vaccines which use Salmonella C500 as carrier.
authors.affiliation: College of Animal Science and Veterinary Medicine, Sichuan Agricultural University, Ya'an 625014, China.
authors.forename: Dishi
authors.initials: D
authors.lastname: Chen
authors.forename: Wanzhu
authors.initials: W
authors.lastname: Guo
authors.forename: Zhiwen
authors.initials: Z
authors.lastname: Xu
authors.forename: Yang
authors.initials: Y
authors.lastname: Chen
authors.forename: Wen
authors.initials: W
authors.lastname: Li
authors.forename: Yin
authors.initials: Y
authors.lastname: Wang
authors.forename: Ling
authors.initials: L
authors.lastname: Zhu
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Wang
mesh_terms: D000818:Animals; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D022562:Salmonella Vaccines / Q000235:genetics / Q000276:immunology; D012476:Salmonella arizonae / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D013552:Swine; D019444:Vaccines, DNA / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D022562:Salmonella Vaccines; D019444:Vaccines, DNA; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins