猪戊型肝炎病毒基因ORF2片段AG02的融合表达及间接ELISA方法的初步建立

采用巢式RT-PCR技术扩增猪戊型肝炎病毒（SHEV）结构蛋白基因ORF2部分片段AG02。将该片段插入pET32a表达载体，命名为PET-HEV，将重组表达质粒转化大肠杆菌BL21（DE-3），经1mM IPTG诱导得到表达，进行SDS-PAGE分析，证明该片段得到融合表达，分子量为33KD。Western blot分析表明，重组蛋白可以与HEV阳性血清反应，表明该蛋白具有良好的抗原性。以纯化的重组蛋白建立了初步的间接ELISA方法,经方阵滴定确定最佳包被浓度为2.43μg/mL，血清最佳的稀释比为1:40,与万泰试剂盒相比较，证明我们建立的ELISA方法有较高的特异性和敏感性。

Cloning and expression of a fragment of swine HEV ORF2 AG02 in E.coli and the Development of Indirect ELISA with

Nest RT-PCR was used to amplified swine hepatitis E virus(SHEV) ORF2-AG02 fragment.AG02 was inserted into pET32a vector and the recombinant expression plasmid PET-HEV was constructed. PET-HEV was transferred into BL21(DE3) and induced by 1mM IPTG, the SDS-PAGE result showed that the 33kDa recombinant protein was expressed. The Western blot analysis showed that the recombinant protein has a good antigenicity with positive SHEV serum. An indirected ELISA was established with the purified recombinant protein, after the square matrix titrate determination best density is 2.43μg/mL, the blood serum best dilution ratio is 1:40,Compares with ten reagents boxes, proved we establish the ELISA method has the high specificity and the sensitivity.