猪戊型肝炎病毒Ⅳ型衣壳蛋白单克隆抗体的制备与鉴定  

Preparation and Characterization of Monoclonal Antibodies Specific for Capsid Protein of Swine Hepatitis E Virus Genotype Ⅳ

作　　者：赵菲菲[1] 赵钦[2] 胡守彬[1] 陈福勇[3] 肖一红[1] 周恩民[2] 

机构地区：[1]山东农业大学动物科技学院免疫生物学实验室,泰安271018 [2]西北农林科技大学兽医免疫学研究所,西北农林科技大学动物医学院,杨凌712100 [3]中国农业大学动物医学院,北京100193

出　　处：《畜牧兽医学报》2012年第6期950-955,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：山东省"泰山学者"建设工程项目(040-72010)

摘　　要：为获得猪戊型肝炎病毒(Hepatitis E virus,HEV)Ⅳ型衣壳蛋白单克隆抗体,将猪HEV衣壳蛋白的C端267(408—675)个氨基酸基因序列克隆入原核表达载体pET-28a(+),构建重组质粒pET-28a-ORF2-C,转化E.coli Rosetta(BL21)感受态细胞进行诱导表达,SDS-PAGE和Western blot鉴定,纯化后免疫小鼠。取免疫小鼠的脾脏与鼠骨髓瘤细胞SP2/0融合制备单克隆抗体。通过间接ELISA和竞争ELISA方法筛选并鉴定单抗。结果表明蛋白得到正确、高效表达,获得3株识别不同的抗原表位区的单克隆抗体,分别命名为Mab-1E4(IgG1)、Mab-2C7(IgG1)和Mab-2G9(IgG2b),其中1E4和2G9能阻断临床阳性猪血清,提示该2株单克隆抗体识别的抗原表位是猪HEVⅣ型衣壳蛋白上重要的抗原表位区,而单抗Mab-2C7不能阻断。本研究为猪HEVⅣ型的诊断及研究提供重要工具。To prepare monoclonal antibodies （Mabs） against capsid protein of swine hepatitis E vi- rus IV （HEV）, the gene of swine HEV encoding C-terminal 267 （408-675） amino acids of capsid protein was cloned into pET-28a （＋） vector to construct recombinant plasmid pET-28a-ORF2-C. The protein was expressed in E. coli Rosetta （BL21）, and identified by SDS-PAGE and Western blot. Spleens of BALB/e mice immunized with the purified protein were used to produce mono- elonal antibodies （Mabs）. The Mabs were selected and characterized with the indirect ELISA and competitive ELISA. Protein was expressed correctly. Three Mabs, designated Mab-2C7, 209 and 1E4, were selected and recognized three independent epitopes. Mab-2G9 is IgG2b and Mab- 2C7 and Mab-lE4 are IgG1. The positive HEV swine sera could be blocked by Mab-lE4 and Mab-2G9 indicating the epitopes recognized by Mab-lE4 and Mab-2G9 are predominant epitopes in capsid protein of swine HEV genotype IVwhile Mab-2C7 can not. The results suggest that theMab-lE4 and Mab-2G9 are potential diagnostic reagents for detection of swine HEV infection genotype Ⅳ.

关 键 词：猪戊型肝炎病毒 衣壳蛋白 单克隆抗体 鉴定 

分 类 号：S852.659.6[农业科学—基础兽医学]