沙门氏菌胞周蛋白二硫键异构酶DsbA对乙肝疫苗HBsAg‘a’表位构象的作用研究

Effect on conformational formation of HBV vaccine 'a' epitope with the assistance of Salmonella typhimurium periplasmic protein disulfide isomerase DsbA

目的:研究沙门氏菌胞周蛋白二硫键异构酶DsbA对乙肝疫苗HBsAg‘a’表位构象的形成作用。方法:将沙门氏菌胞周蛋白二硫键异构酶DsbA基因克隆于PET32b载体上,与乙肝疫苗HBsAg‘a’表位载体共转BL21菌,运用流式细胞仪和荧光显微镜观察HBsAg‘a’表位构象的形成。结果:在没有沙门氏菌胞周蛋白二硫键异构酶DsbA的情况下,HBsAg‘a’表位的正确表达率仅为29.73%,而在共转沙门氏菌胞周蛋白二硫键异构酶DsbA的BL21菌中,HBsAg‘a’表位的正确表达率上升至59.78%,较前提高了1倍。荧光显微镜观察发现加入β-二巯基乙醇破坏二硫键的形成后,DsbA辅助形成HBsAg‘a’表位的绿色荧光也随之消失。结论:沙门氏菌胞周蛋白二硫键异构酶DsbA能够有效促进原核细菌胞膜上HBsAg‘a’表位构象的正确形成。

Objective： To study the effect on conformational formation of HBV vaccine ＇a＇ epitope by Salmonella typhimurium periplasmic protein disulfide isomerase DsbA.Method： The ORF of Salmonella typhimurium periplasmic protein disulfide isomerase DsbA was cloned into PET32b vector which was co-transferred with HBsAg ＇a＇ epitope plasmid into BL21 competence.The conformational epitope formation of HBsAg ＇a＇ were observed by flow cytometry and fluorescence microscopy.Results： The frequency of correct conformational epitope formation of HBsAg ＇a＇ epitope without Salmonella typhimurium periplasmic protein disulfide isomerase DsbA is only 29.73%,but it is up to 59.78% with co-transfected Salmonella typhimurium periplasmic protein disulfide isomerase DsbA,1-fold compared with the former.After 2-mercaptoethanal was added into the medium to destroy disulfide bonds,the green fluorescent of FITC of HBsAg ＇a＇ epitope disappeared subsequently in visual field of fluorescence microscopy.Conclusion： Salmonella typhimurium periplasmic protein disulfide isomerase DsbA can effectively promote the correct conformation forming of HBsAg ＇a＇ epitope on the membrane of prokaryotic bacteria.