摘要
利用RT-PCR方法扩增出IPNV-ZYX分离株主要结构蛋白VP2的抗原表位区基因(616bp),命名为IPNV VP2 COE,将其克隆到pCold TF表达载体中构建重组质粒pCold TF-VP2COE,在大肠杆菌BL21(DH5α)感受态表达,经SDS-PAGE电泳分析,表达蛋白约78 ku,用镍离子亲和层析柱纯化该蛋白,制备抗血清,间接ELISA结果显示,IPNV(ATCC VR-1318)细胞培养物与鼠抗VP2 COE蛋白血清发生特异性反应,效价为1∶12 800;间接免疫荧光结果显示,鼠抗VP2 COE血清可与黑龙江某渔场已知感染IPNV虹鳟肝组织产生特异性的荧光,以上两项结果表明,表达IPNV VP2 COE蛋白具有良好的免疫原性和免疫反应性,为IPNV检测方法的建立及疫苗的制备提供理论依据。
Infectious pancreatic necrosis virus （IPNV） is a major viral pathogen of salmonid fish and causes serious economic losses to salmonid aquaculture. We amplified a 616 bp epitope of the VP2 gene from recent IPNV-ZYX isolated from fanned rainbow trout （Oncorhynchus mykiss） of China, and cloned into pCold TF vector（designated as pCold TF-VP2 COE）. The expression of recombinant plasmid pCold TF-VP2 COE in E. coli BL21（DE3） was induced and detected by SDS-PAGE analysis. The predicted molecular weight for recombinant VP2 COE protein was approximately 78 ku and this was confirmed in this study. The fusion protein was purified with ProBondTM resin from the suspension centrifuged and the an- tisera against VP2 COE protein were produced. The prepared antisera reacted specifically with IPNV（ATCC VR-1318）antigen by indirect ELISA. The antisera against VP2 COE protein had OD values at least twice that obtained for the negative control serum at a dilution of 1：12 800. IFA showed specific reaction of the antisera against VP2 COE protein with liver samples of rainbow trout naturally infected with IPNV in Heilongjiang province. The results showed that the expressed VP2 COE protein was immu- nogenical and antigenical which was the same as the natural IPNV VP2 protein. All this work established a foundation for further study on vaccine and rapid diagnosis of IPNV.
出处
《水产学报》
CAS
CSCD
北大核心
2012年第11期1770-1775,共6页
Journal of Fisheries of China
基金
国家科技支撑计划(2012BAD25B02)
黑龙江省教育厅科技项目(11541019)
