论文部分内容阅读
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 coat protein (GCRV-VP5) gene of GCRV-VP5 was amplified from the grass carp Ctenopharyngodon idellus hemorrhagic disease-GCRV-873 strain. The GCRV-VP5 gene was cloned into the prokaryotic expression vector pET-30α and transformed into Escherichia coli E.coli BL-21 (DE3) and induced with IPTG to obtain the total protein with the recombinant plasmid. The antiserum obtained from the goat immunized with GCRV-873 strain was used as the primary antibody to perform western blotting. As a result, a specific immunoreactive band with a relative molecular mass of 77,000 was detected on the nitrocellulose membrane, which was identical to the predicted GCRV-873 VP5 protein, suggesting that E. coli fusion expression of grass carp hemorrhagic disease virus VP5 protein. The expressed fusion protein mainly exists as an insoluble inclusion body, and the protein purity can reach 90% after being purified by column chromatography. The fusion protein obtained in this study can provide antigens for the later immunization animals, and provide the basis for establishing the immunological detection method of GCRV.