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

【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。

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

【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.