传染性胰腺坏死病毒VP2 COE蛋白单克隆抗体的制备与初步应用  

Preparation and preliminary application of monoclonal antibodies against VP2 COE protein of infectious pancreatic necrosis virus

作　　者：连科迅[1] 赵丽丽[1] 张琳琳[2] 贾鹏[3] 唐立杰[1] 葛俊伟[1] 李一经[1] 刘敏[2] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]东北农业大学动物科学与技术学院,黑龙江哈尔滨150030 [3]深圳出入境检验检疫局,广东深圳518045

出　　处：《水产学报》2013年第8期1229-1235,共7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)

摘　　要：为制备抗IPNV VP2蛋白的单克隆抗体,对其基本特性进行鉴定并进行初步应用。实验利用Ni-NTA亲和层析纯化的IPNV VP2 COE重组蛋白作为免疫原,免疫8周龄的雌性BALB/c小鼠,经3次免疫后,将免疫小鼠的脾细胞与骨髓瘤SP2/0细胞融合。采用间接ELISA和有限稀释法筛选杂交瘤细胞,共得到2株能稳定分泌特异性抗体的阳性细胞株,分别命名为5G10和5F3,亚类鉴定均为IgG1亚类。2株杂交瘤细胞的染色体数目在75～120之间。间接ELISA检测5G10和5F3细胞培养上清的效价分别为1∶105、1∶102,腹水效价分别为1∶108、1∶104。Western-blotting和间接免疫荧光鉴定结果显示,2株单抗均能特异性地识别IPNV。间接ELISA表明,2株单抗不与IHNV、VHSV、SVCV、HRV等病毒反应,说明获得的单抗具有高度的特异性。相加ELISA实验结果显示,2株单克隆抗体分别识别IPNV VP2蛋白上不同的抗原位点。应用间接免疫荧光方法对临床确定为患有IPN虹鳟肝组织进行检测,结果证实该2株单克隆抗体可用于后续实验。The recombinant protein IPNV VP2 was used as immunogen after purification by Ni-NTA. The 8- week-old BALB/c mice were intraperitoneally immunized with the VP2 protein for three times, then myeloma cells SP2/0 were fused with the spleen cells of the immunized BALB/c mice. Two hybridoma cell lines against the VP2 protein were obtained by screening with the indirect ELISA and limiting dilution assay, which were identified to be lgG1 subtype and named 5G10,5F3. The numbers of chromosomes of the two hybridomas were in the range of 75 to 120. Their antibody titers of cells culture supernatant were 1 ： 10^5 , 1 ： 10^2 respectively. Their titers of ascites were 1 ： 10^8 , 1 ： 10^4 respectively. Western-blot analysis and indirect immunofluorescence showed that the two McAbs could react with IPNV specificity. The 2 McAbs had no reactive capability with IHNV, VHSV, SVCV and HRV by the indirect ELISA. Antibodies additivity assay demonstrated that 5G10 and 5F3 recognized the different epitopes of IPNV VP2 nucleoprotein. We detected the clinical suffering from IPN rainbow trout liver tissue material, and the results confirmed that the 2 McAbs can be used for follow-up testing.

关 键 词：传染性胰腺坏死病毒 VP2重组蛋白 单克隆抗体 

分 类 号：Q785[生物学—分子生物学] S941.41[农业科学—水产养殖]