草鱼呼肠孤病毒Ⅱ型VP6蛋白多克隆抗体制备及其特异性分析

Preparation and specification analysis of polyclonal antibody against grass carp reovirus type Ⅱ VP6 protein

为了制备高效的草鱼呼肠孤病毒Ⅱ型VP6蛋白多克隆抗体并对其特异性进行鉴定,实验以草鱼呼肠孤病毒Ⅱ型HZ08株为模板,采用PCR方法扩增S9基因,将该S9基因与p ET-32a(+)载体连接构建p ET-32a-S9原核表达载体,转化到大肠杆菌BL21(DE3)后用IPTG诱导表达;纯化后的重组VP6蛋白免疫新西兰兔,制备多克隆抗体,使用间接ELISA方法测定抗体效价,Western blot和IFA试验鉴定抗VP6蛋白多克隆抗体特异性。结果显示草鱼呼肠孤病毒Ⅱ型的S9基因在原核表达载体中能够正确地表达VP6蛋白,纯化的重组蛋白免疫新西兰兔制备的抗VP6多克隆抗体,经间接ELISA方法测定其效价约为1∶105,Western blot和IFA试验结果显示制备的多克隆抗体能特异性识别GCRVⅡ型毒株,而不能识别GCRV I型、Ⅲ型以及其它病毒,表明该多克隆抗体具有较高的特异性。研究表明制备的抗VP6蛋白多克隆抗体能够特异性识别GCRVⅡ型病毒,为GCRVⅡ型病原学研究及草鱼出血病临床诊断奠定基础。

In order to prepare the efficient anti-VP6 polyclonal antibody of grass carp reovirus genotype II(GCRV-II)and identify its specificity,grass carp reovirus genotype II strain HZ08 was used as the template to amplify the S9 gene of grass carp reovirus type II by PCR method.The S9 gene fragment was ligated with pET-32a(+)vector to construct the pET-32a-S9 prokaryotic expression vector.It was transformed to E.coli BL21(DE3)for expression,being inducted by IPTG.New Zealand rabbits were injected with the purified recombinant protein for immunization.The titer of the prepared polyclonal antibody was determined by indirect ELISA.And the specificity of antibody was determined by Western blot and indirect immunofluorescence assay(IFA).The results showed that the VP6 protein encoded by the S9 gene that can be expressed correctly in prokaryotic expression vector.The titer of the polyclonal antibody was detected to be 1∶10 5,by using the indirect ELISA method.The results of Western blot and IFA showed that the polyclonal antibody could recognize the GCRV type II strain with high specificity,but could not identify GCRV type I andⅢ,or either other viruses.The polyclonal antibody against VP6 protein could specifically identify GCRV type II strain.In summary,this study laid the foundation for researching on the specificity of GCRV type II and the clinical detection of grass carp hemorrhage disease.