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

Cloning and Expression of a Fragment of Swine HEV ORF2 AG02 in E.coli and the Development of Indirect ELISA with Recombinant Protein

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1mmol/LIPTG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33kDa。Westernblot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。建立的ELISA方法有较高的特异性和敏感性。

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-HEY was transferred into BL21 （DE3） and induced by 1mmoL/L 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 the ELISA method has the high specificity and the sensitivity.