Hsp65与HBV多表位融合基因的表达及其免疫应答研究  

Expression and antigenicity identification of the fusion protein of Hsp65 and hepatitis B virus multi-epitope

作　　者：王华[1] 尹晓光[2] 张培因[1] 卫红飞[1] 于永利[2] 王丽颖[1] 

机构地区：[1]吉林大学基础医学院分子生物学教研室,长春130021 [2]吉林大学基础医学院免疫学教研室,长春130021

出　　处：《免疫学杂志》2008年第5期487-490,共4页Immunological Journal

基　　金：国家自然科学基金海外杰出青年基金项目(30328010)

摘　　要：目的用基因工程的方法构建并表达一种由Hsp65、通用辅助T细胞表位(PADRE)和HBV表面抗原与核心抗原的多个表位组成的乙型肝炎治疗性疫苗(简称为Hsp65-HBV)。方法采用PCR方法人工合成由PADRE和HBV的多个表位组成的基因片段,将此片段克隆入原核表达质粒pET28a/Hsp65后,利用大肠杆菌进行融合蛋白的表达。用纯化后的蛋白免疫Balb/c小鼠。将小鼠脾细胞用HBsAg装载的P815细胞在体外刺激5d后,用流式细胞仪检测IFN-γ+细胞的频率。用ELISA方法检测小鼠血清中的特异性抗体(IgG1和IgG2a)的产生。结果由Hsp65、通用辅助T细胞表位(PADRE)和HBV的多个表位组成的融合蛋白能在大肠杆菌中进行高水平的表达,能诱导小鼠产生IgG2a亚型的特异性抗体,并能诱导HBV特异性的IFN-γ+的淋巴细胞的产生。结论获得了大肠杆菌表达的由Hsp65、通用辅助T细胞表位(PADRE)和HBV的多个表位组成的融合蛋白,并能被机体以MHC-Ⅰ途径递呈,为检测本融合蛋白是否能激发出HBV特异性CTL反应奠定了基础。Objective To construct a fusion protein containing Hsp65 and HBV epitopes which can stimulate the specific cellular immune against HBV. Methods HBV multi-epitope and universal helper T cell epitope （PADRE） were fused to the downstream of BCG heat shock protein 65 （in short, Hsp65-HBV）. The fusion protein was expressed in E. coli and used to immunize Balb/c mice. Single spleen cell suspensions were stimulated with HBsAg-pulsed P815 cells. The IFN-γ-producing cell subsets were determined and analyzed by FACS. The specific antibodies of IgG1 and IgG2a subclasses induced by Hsp65-HBV were also detected. Results Fusion protein of Hsp65-HBV was expressed and could induce the production of HBV-specific IFN-γ-producing cells and the specific IgG2a subclass of antibodies. Coneulsion Fusion protein of Hsp65-HBV is obtained and can be presented by MHC class Ⅰ pathway, which would provide a base for detecting the CTL reaction stimulated by the hepatitis B vaccine.

关 键 词：热休克蛋白65 PADRE 乙型肝炎病毒 多表位 

分 类 号：R392[医药卫生—免疫学]