草鱼呼肠孤病毒VP5蛋白的表达与纯化

Expression and purification of VP5 protein in grass carp reovirus

从草鱼Ctenopharyngodon idellus出血病病原——草鱼呼肠孤病毒GCRV-873株扩增病毒的外壳蛋白(GCRV-VP5)基因,并将GCRV-VP5基因克隆到原核表达载体pET-30α,转化至大肠杆菌E.coli BL-21(DE3)中后,用IPTG诱导培养,获得带有重组质粒菌体的总蛋白。用GCRV-873毒株免疫山羊得到的抗血清作为一抗进行免疫印迹试验,结果在硝酸纤维素膜上检测到在相对分子质量为77 000处有特异性免疫条带,与预测的GCRV-873 VP5蛋白一致,提示大肠杆菌融合表达草鱼出血病病毒的VP5蛋白。表达的融合蛋白主要以不可溶的包涵体形式存在,经柱层析纯化后蛋白纯度可达90%。本试验中获得的融合蛋白可为后期免疫动物提供了抗原,也为建立GCRV免疫学检测方法提供了依据。

The GCRV-VP5 gene of grass carp reovirus （GCRV） was amplified from GCRV- 873 strain, and cloned into plasmid pET-30α which was transformed into bacterium Escherichia coli BL-21 （ DE3 ） from which the total protein was obtained by IPTG. The Western bloting using goat anti-GCRV serum showed a 77 000 specific band, indicating that the transformed E. coli express GCRV-VP5 protein. The recombinant proteins were expressed in E. coli in the form of inclusion bodies and purified with purity level of over 90%. The findings provide fused pro- tein as an antigen to immunize animals.