OmpC-HBsAg‘a’表位嵌合蛋白疫苗的构建表达及其对HBV转基因小鼠的免疫效果研究

Construction and expression of OmpC-HBsAg 'a' epitope chimeric protein vaccine and study of its immune effect on HBV transgenic mice

目的：构建OmpC-HBsAg‘a’表位嵌合蛋白疫苗来研究对HBV转基因小鼠的免疫效果。方法：将HBsAg‘a’抗原决定簇主要肽段S-（124-147aa）插入到沙门氏菌外膜孔道蛋白OmpC第4 Loop区,并运用DsbA进行辅助二硫键形成,对HBV转基因小鼠免疫,激活固有免疫系统中的B-1细胞产生HBsAb。结果：HBsAg的adr‘a’表位CTSPAQGTSMF-PSCCCTKPSDGNC成功插入OmpC-HBsAg‘a’基因的588~659 bp之间,重组后的载体PCR鉴定和测序结果完全与设计相一致。IPTG诱导表达后,荧光显微镜显示3E7抗体能够识别BL21外膜表面的OmpC-HBsAg‘a’表位嵌合蛋白,用表达该蛋白的BL21直接免疫HBV转基因小鼠,能够产生HBsAb抗体。结论：OmpC-HBsAg‘a’表位嵌合蛋白在原核中表达具有天然构象,以此蛋白为基础的疫苗能够打破HBV转基因小鼠对HBsAg的免疫耐受。

Objective： To study the immune effect of OmpC-HBsAg ＇a＇ epitope chimeric protein vaccine on HBV transgenic mice.Methods： The major peptide of antigenic determinant from HBsAg ＇a＇,S-（124-147aa）,was inserted into the fourth Loop of OmpC,the outer membrane pore protein of Salmonella,then disulfide bond was formed by the assistant of DsbA.When HBV transgenic mice were immunized with OmpC-HBsAg ＇a＇ epitope chimeric protein vaccine,B-1 cells were activated and secreted HBsAb in their native immune system.Results： adr ＇a＇ epitope of HBsAg,CTSPAQGTSMFPSCCCTKPSDGNC,was successfully inserted between 588 bp and 659 bp of OmpC-HBsAg ＇a＇ gene,which was identified by PCR and sequencing result of which was entirely consistent with the design.After the induction of IPTG,3E7 antibodies were able to recognize the OmpC-HBsAg＇a＇ epitope chimeric protein on the outer membrane of BL21,which could be detected by fluorescent microscopy.HBV transgenic mice generated HBsAb when directly immunized with BL21 which were expressing this protein.Conclusion： The expression of OmpC-HBsAg＇a＇ epitope chimeric protein in prokaryotic cells has natural conformation.The vaccine based on this protein can break the immune tolerance to HBsAg in HBV transgenic mice.