蓝舌病病毒NS1蛋白的截短表达、纯化及其多克隆抗体制备

Expression,Purifi cation and Antibody Preparation of Truncated NS1 Protein of Bluetongue Virus

本研究通过对蓝舌病病毒1型(BTV1)的NS1基因进行抗原性分析,选取其中抗原指数较高的片段(1~960 bp),利用RT-PCR扩增获得NS1截短基因序列,插入原核表达载体pET-30a中构建重组表达质粒pET-NS1-320aa,将该重组质粒转入大肠杆菌BL21感受态细胞,经IPTG诱导表达并进行可溶性分析,利用SDS-PAGE对截短NS1蛋白的表达进行检测,发现重组NS1蛋白主要以包涵体的形式存在于菌体中。利用组氨酸标签纯化树脂获得了高纯度的NS1蛋白;将其免疫新西兰大白兔制备的抗NS1多克隆抗体不仅可与重组表达的截短NS1蛋白反应,而且可与BTV1感染细胞中的天然NS1蛋白发生特异性反应;免疫荧光实验发现在BTV1感染的BHK-21细胞中检测到了NS1蛋白的特异表达,且分布于细胞质中。本研究成功表达了BTV重组NS1截短蛋白并制备了相应的特异性抗体,为研究NS1蛋白的特性和功能奠定了基础。

In this study,the truncated NS1 gene sequence with higher antigenic index(1-960 bp)of Bluetongue virus type 1(BTV1)was obtained by RT-PCR amplification and inserted into the prokaryotic expression vector pET-30a to construct the recombinant expression plasmid pET-NS1-320aa.The recombinant expression plasmid was transformed into E.coli BL21 competent cells for expression with IPTG.The expressed truncated NS1 protein was detected by SDS-PAGE.The recombinant NS1 protein mainly existed in the form of inclusion bodies.The highly purified NS1 protein was obtained using histidine tag purification resin.The prepared anti-NS1 polyclonal antibodies in New Zealand rabbits reacted with the truncated NS1 protein and detected BTV1 in infected cells.In addition,NS1 protein was detected in BHK21 cells infected with BTV1 and distributed throughout the cytoplasms.The availability of the truncated NS1 protein of BTV and specific antibodies laid a foundation for further studying the characteristics and functions of NS1 protein.