猪戊型肝炎病毒ORF2基因的分段原核表达及抗原表位的初步鉴定

Prokaryotic Expression and Preliminary Identification of Antigenic Epitope of the Different Fragments of ORF2 Gene of Swine Hepatitis E Virus

为了初步鉴定猪戊型肝炎病毒（sw HEV）ORF2蛋白的抗原表位,通过设计两对引物,PCR扩增ORF2-1和ORF2-2两段基因片段,使用p MD18-T载体克隆,双酶切后构建重组质粒p ET32a-0RF2-（1,2）,转化大肠杆菌BL21后进行IPTG诱导和SDS-PAGE凝胶电泳,最后用4株ORF2单抗对其进行Western Blot来初步鉴定其抗原表位。结果表明,分别获得约为36 ku和35 ku的两个目的蛋白,均以不可溶形式存在,抗原表位初步定位于ORF2-1这段蛋白中的414~523 aa。

To identify the antigenic epitope of the ORF2 protein of swine hepatitis E virus,the ORF2-1 and ORF2-2 frag-ments were respectively amplified by designing two pairs of specific primers.The two fragments were cloned into p MD18-T vector.The recombinant plasmids p ET32a-ORF2-1,2 were constructed and transformed into E.coli BL21.After IPTG induction,the pro-tein was analyzed by SDS-PAGE and Western-blot. Its antigenic epitope of the ORF2 was preliminarily identified by four ORF2 monoclonal antibodies.The result showed that the two target proteins were obtained successfully and they were 36 ku and 35 ku re-spectively.Its antigenic epitope was preliminarily identified as the 414~523 aa of the ORF2-1 protein.