猪戊型肝炎病毒ORF2片段克隆及原核表达

Cloning and expression of a fragment of swine HEV ORF2 in E.coli

设计套式引物,内套引物含有BamHⅠ/XhoⅡ酶切位点,采用RT-PCR技术扩增猪戊型肝炎病毒(HEV)结构蛋白基因ORF2部分片段,长度为634bp,将其克隆于PGEX-T载体,测序结果表明插入的片段属于戊型肝炎病毒ORF2部分,将该片段插入PproEXHTb表达载体,经酶切鉴定证实获得了重组表达质粒。将重组质粒转化大肠杆菌BL21,经1mmol/LIPTG诱导,得到表达,表达蛋白分子量为27Ku,以包涵体形式存在。Westernblot分析表明,该蛋白可以与猪戊型肝炎阳性血清反应,表明该蛋白具有良好的反应原性。

By designing a pair of primers for nest -PCR, which have Barn H Ⅰ/Xho Ⅱ site, a fragment of swine HEV ORF2 was applied by nest RT-PCR and cloned into the vector PGEX-T. Alter sequencing, the result showed that the insert fragment was a part of ORF2 of swine HEV. The fragment was inserted into the bacterial plasmid PproExhTb and the recombinant plasmid was identified with restriction enzyme analysis. After induced with l mmol/L IPTG, the protein was expressed in bacterial BL21. The result showed that the fragment was expressed as the form of deposit, the protein was 27 Ku. The result of western blot analysis dictated that the protein could react with the HEV positive serum.