猪伪狂犬病病毒gE蛋白单克隆抗体的制备及胶体金免疫层析检测方法的建立

Preparation of monoclonal antibody against gE protein of Pseudorabies virus and establishment of colloidal gold immunochromatographic assay

为了建立一种简单、快速检测猪伪狂犬病病毒(PRV)野毒株的方法,试验原核表达gE蛋白,鉴定并纯化后免疫Balb/c小鼠,采集免疫Balb/c小鼠的脾脏进行细胞融合,筛选单克隆杂交瘤细胞株扩大培养。将阳性杂交瘤细胞免疫Balb/c雌性小鼠,小鼠腹部明显膨大后采集腹水,利用间接ELISA方法测定抗体效价;采用秋水仙素法对筛选到的阳性杂交瘤细胞进行染色体数目测定;利用SBAClonotyping^(TM)System/HRP亚类鉴定试剂盒鉴定单克隆抗体亚类;利用Western-blot方法鉴定单克隆抗体活性;利用得到的单克隆抗体制备胶体金免疫层析试纸条,考察胶体金免疫层析试纸条的特异性、敏感性、重复性,进行符合性试验、临床应用试验。结果表明:获得了以包涵体形式表达的重组gE蛋白,Western-blot检测具有良好的免疫学活性;以重组gE蛋白为免疫原制备了3株抗PRV gE蛋白单克隆抗体4C8、5D10、6E8,其注射小鼠后产生的腹水抗体效价分别为1∶512 000、1∶1 024 000、1∶512 000,Western-blot检测均具有良好的免疫学活性;杂交瘤细胞4C8、5D10、6E8的染色体数分别为1×10^(5)、1×10^(6)、1×10^(6),均接近脾细胞和骨髓瘤细胞染色体数的总和。分别以5D10和6E8为标记抗体和检测抗体,组装了检测PRV野毒株的胶体金免疫层析试纸条,用试纸条检测PRV疫苗株、PPV、PCV-2、CSFV、PRRSV、TGEV的结果均为阴性,对PRV野毒株的最低检出量为1×10^(2.83) TCID_(50),批内和批间重复检测的结果完全相同,与PCR方法的符合率达92.59%,检测157份临床病料样品的阳性率为17.83%。说明胶体金免疫层析试纸条具有良好的特异性、敏感性、重复性,适合基层养殖单位进行PRV野毒的快速鉴别诊断。

In order to establish a simple and rapid method for detection of Pseudorabies virus(PRV) wild strain, The test prokaryotic expressed gE protein, identified and purified, then immunized Balb/c mice, collected the spleen of Balb/c mice for cell fusion, screened monoclonal hybridoma cell lines and expanded culture. Balb/c female mice were immunized with positive hybridoma cells, ascites was collected after abdominal expansion, the antibody titer was determined by indirect ELISA. The chromosome number of positive hybridoma cells was determined by colchicine method. Monoclonal antibody subclasses were identified by SBAClonotyping^(TM)System/HRP subclass identification kit. The activity of monoclonal antibody was identified by Western-blot. Colloidal gold immunochromatographic strip was prepared by using the obtained monoclonal antibody, and to investigate the specificity, sensitivity and repeatability of colloidal gold immunochromatographic test strip, and carry out compliance test and clinical application test. The results showed that the recombinant gE protein expressed in the form of inclusion body was obtained, and had good immunological activity by Western-blot;three monoclonal antibodies 4 C8, 5 D10, 6 E8 against PRV gE protein were prepared using recombinant gE protein as immunogen, and the ascites antibody titer produced after injection into mice were 1∶512 000,1∶1 024 000,1∶512 000, which had good immunological activity by Western-blot. The chromosome numbers of hybridoma cells 4 C8, 5 D10, 6 E8 were 1×10^(5)、1×10^(6)、1×10^(6), which are very close to the sum of chromosome numbers of splenocytes and myeloma cells. Using 5 D10 and 6 E8 as labeled antibody and detection antibody respectively, a colloidal gold immunochromatographic strip for detection of PRV wild strain was assembled;the detection of PRV vaccine strain, PPV, PCV-2, CSFV, PRRSV and TGEV was negative using the strip;the minimum detectable amount for PRV wild strain was 1×10^(2.83) TCID_(50). The results of intra assay and inter assay were identical, and the coincidence rate with PCR was 92.59%. The positive rate of 157 clinical samples was 17.83%. The results suggested that the colloidal gold immunochromatographic strip had good specificity, sensitivity and repeatability, which was suitable for the rapid differential diagnosis of PRV in primary breeding units.