猪戊型肝炎病毒ORF2主要抗原表位区间接ELISA方法的初步建立

应用原核表达系统表达猪戊型肝炎病毒ORF2蛋白C端和N端的主要抗原表位区，重组蛋白命名为ORF2-C和ORF2-N，初步建立间接ELISA诊断方法。用重组蛋白ORF2-C和ORF2-N作为诊断抗原，对反应条件进行优化，初步建立ELISA诊断方法。抗原最适包被浓度ORF2-C为8 μg/mL、ORF2-N为12 μg/mL；血清最适稀释度均为1∶50，ORF2-C作用时间为50 min、ORF2-N作用时间为90 min；酶标抗体最适稀释度为1∶5000；ORF2-C判定标准：D450 nm值≥0.348为阳性，D450 nm值<0.348为阴性；ORF2-N判定标准：D450 nm值≥0.397为阳性，D450 nm值<0.397为阴性。与戊型肝炎病毒诊断试剂盒检测结果相比，阳性符合率分别为93.3%、86.7%。利用重组蛋白建立的ELISA方法特异性、敏感性和重复性均较好，且ORF2-C的检测效率明显高于ORF2-N，该方法的初步建立为进一步完善猪戊型肝炎病毒诊断方法奠定基础。

Development of Indirect ELISA with Major Epitope Domain of ORF2 of SHEV

C-terminal and N-terminal major epitope domain of ORF2 of swine Hepatitis E (SHEV) were express in E.coli, denominated ORF2-C and ORF2-N respectively. Indirect ELISA assay were developed to detect antibody of swine hepatitis E virus using recombinant proteins. Recombinant proteins were used as diagnostic antigen. Indirect ELISA assay were develop for detect antibody of SHEV. This assay was optimized for ORF2-C and ORF2-N antigen coating concentration of 8 and 12 μg/mL respectively and a serum dilution of 1∶50, with a standard incubation time of 50 and 90 min respectively. ORF2-C a reading of D450 nm≥0.348 and ORF2-N a reading of D450 nm≥0.397 was scored as positive. The positive coincident rate between the fusion protein ELISA and the available Kit was up to 93.3 % and 86.7% respectively in detecting swine HEV antibody. The result showed the indirects ELISA had a better sensitivity, specificity and repeatability. ORF2-C was significantly better than the ORF2-N used as coating antigen. This research was beneficial to further development of diagnostic kit for swine hepatitis E.