鸽源新城疫病毒F基因的克隆与原核表达

Cloning and prokaryotic expression of F gene of Newcastle disease virus from pigeon

采用RT-PCR方法扩增了鸽源新城疫病毒F基因并将其克隆至pMD18-T载体,测定其核苷酸序列。根据F基因CDS序列,设计原核表达引物,扩增F基因功能区片段,并构建了重组原核表达质粒pGEX4T-1-NDV F(924),在不同时间和温度下进行诱导表达。通过SDS-PAGE检测,目的基因表达出约60ku的融合蛋白,主要以包涵体形式存在,表达目的蛋白的最佳诱导时间为6h,最佳诱导温度为42℃;提取并纯化包涵体蛋白,用其免疫BALB/c小鼠,采小鼠血进行ELISA检测。结果显示,表达蛋白具有良好的免疫原性和反应原性。上述结果表明,缺失疏水区的F基因可以在大肠杆菌BL21(DE3)中获得表达,表达的重组F蛋白具有良好的免疫原性和反应原性。

The F gene was amplified from Newcastle disease virus Gansu strain from a pigeon by RTPCR.The PCR product was cloned into pMD18-T vector,and the recombinant plasmid was sequenced.According to the CDS sequence of F gene,the primers were designed for expression of F protein.The fragment of F gene was amplified with the primers by PCR,then it was cloned into pGEX-4T-1vector and expressed in Escherichia coli.The target gene was induced and expressed in different time points and at different temperatures.The fusion protein was mainly existed in the form of inclusion bodies,and its molecular weight was 60ku by SDS-PAGE analysis.The optimum induction time was 6hand the optimum induction temperature was 42℃.BALB/c mice were immunized by the recombinant protein which was extracted and purified from inclusion bodies.The fusion protein had high reactionogenicity and immunogenicity by the indirect ELISA.In conclusion,the F gene,whose the hydrophobic domain sequence was deleted,still could be expressed in E.coli BL21(DE3),and the expressed recombinant F protein had good immunogenicity and reactionogenicity.