蓝舌病病毒NS3蛋白的截短表达及其多克隆抗体制备

Expression of truncated NS3 protein of bluetongue virus and its antibody preparation

首先利用RT-PCR技术扩增获得了蓝舌病病毒1型(BTV1)NS3基因的截短片段,将其克隆至表达载体pET-30a中,构建重组表达质粒pET-NS3,转化大肠杆菌BL21(DE3)感受态细胞后用IPTG诱导表达,可溶性分析显示,重组NS3蛋白在大肠杆菌中以包涵体形式表达。纯化重组表达的NS3蛋白,免疫新西兰白兔制备抗NS3蛋白的多克隆抗体,利用免疫印迹和细胞免疫荧光试验检测该抗体的特异性及NS3蛋白在BTV感染细胞内的分布。结果显示,制备的抗NS3多克隆抗体不仅可与重组表达的NS3蛋白反应,而且可与BTV1感染细胞中的天然NS3/NS3A蛋白发生特异性反应;在BTV1感染的BHK21细胞以及KC细胞中均检测到了NS3/NS3A蛋白的特异性表达,且分布于整个细胞质中。本研究成功表达了BTV重组NS3蛋白并制备了相应的特异性抗体,为进一步研究NS3蛋白的特性和功能奠定了基础。

The truncated NS3 gene of bluetongue virus serotype 1(BTV1)was amplified by reverse transcription-polymerase chain reaction(RT-PCR)and inserted into prokaryotic expression vector p ET-30 a.The recombinant expression plasmid p ET-NS3 was constructed and transformed into Escherichia coli BL21(DE3)competent cells,and then induced with IPTG.Solubility analysis showed that the recombinant NS3(r NS3)protein was expressed as inclusion bodies in E.coli.The r NS3 protein was purified,and was used to immunize New Zealand white rabbits to prepare the polyclonal antibodies.The results of Western-blot showed that the prepared anti-NS3 polyclonal antibody could react with the recombinant NS3 protein as well as the native NS3/NS3 A protein in BTV1 infected cells.Immunofluorescence assay revealed that the NS3/NS3 A proteins were expressed in the whole cytoplasm of BTV-infected BHK21 cells and KC cells.In conclusion,the recombinant NS3 proteins of BTV1 were expressed in E.coli and its polyclonal antibodies were prepared,which will lay a foundation for further revealing the characteristics and functions on NS3 protein.