禽戊型肝炎病毒中国分离株ORF3蛋白的真核表达与抗原性分析  

Eukaryotic Expression and Antigencity Analysis of Avian Hepatitis E Virus ORF3 Protein from China Isolate

作　　者：胡守彬[1] 赵钦[2] 赵菲菲[1] 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预防兽医系免疫生物学实验室,山东泰安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,陕西杨凌712100

出　　处：《中国农业科学》2012年第11期2288-2294,共7页Scientia Agricultura Sinica

基　　金：山东省"泰山学者"建设工程项目(040-72010)

摘　　要：【目的】为获得禽戊型肝炎病毒(hepatitis E virus,HEV)中国分离株(CaHEV)ORF3基因重组蛋白,并对其抗原性进行分析【。方法】提取CaHEV总RNA,通过RT-PCR获得ORF3基因,构建重组质粒pFastBac-HTB-ORF3,转座DH10Bac感受态细胞,提取重组杆粒Bacmid-ORF3,转染sf9细胞,用SDS-PAGE、IFA和Western blot对重组蛋白表达进行鉴定。优化表达条件,将纯化的ORF3蛋白免疫小鼠制备多克隆抗体,用制备的多抗分别与截短表达的3段CaHEV ORF3蛋白进行Western blot和ELISA检测,分析ORF3蛋白的抗原表位区。【结果】SDS-PAGE、Western blot和IFA结果表明CaHEV ORF3蛋白被成功表达且存在细胞内,昆虫细胞在接毒后4天表达量最高。将ORF3重组蛋白免疫小鼠,利用间接ELISA方法,对制备的多抗倍比稀释检测,效价达到104,Western blot和ELISA分析表明ORF3蛋白C端74—88氨基酸处具有较强的抗原性。【结论】本试验用Bac-to-bac系统成功构建含有CaHEV ORF3基因的重组杆状病毒,并在昆虫细胞中得到表达,其主要抗原表位位于C端74—88氨基酸,为进一步研究禽HEV ORF3的蛋白结构和功能奠定了基础。【Objective】The objective of the study is to obtain recombinant protein of avian hepatitis E virus（HEV） ORF3 of China isolate（CaHEV） and analyze its antigencity.【Method】Total RNA was extracted from CaHEV and ORF3 gene was amplified by RT-PCR and cloned into pFastBac-HT.The recombinant plasmid was transformed into DH10Bac and transfected into sf9 insect cells to express the recombinant ORF3 protein.The recombinant ORF3 protein was identified by SDS-PAGE,Western bolt and IFA methods.The purified ORF3 protein was used to immunize Balb/c mice to produce polyclonal antibodies and then to analyze the epitopes in ORF3 using 3 truncated ORF3 proteins 【Result】The results showed that ORF3 protein was expressed with the highest expression level at 4 days post inoculation.The titer of anti-ORF3 polyclonal antibodies was 104determined with the ELISA method.The dominant epitopes of ORF3 were located between amino acids 74 and 88 in C-terminal region.【Conclusion】Recombinant ORF3 protein from avian HEV China isolate was expressed successfully and the dominant epitopes were located between amino acids 74 and 88 in C-terminal region.These results paved a way for future study of the structure and function of ORF3 protein.

关 键 词：禽戊型肝炎病毒中国分离株 ORF3蛋白 真核表达 抗原性 

分 类 号：S852.65[农业科学—基础兽医学]