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

Q4 Environmental Science

水产学报 Pub Date : 2013-01-01 DOI:10.3724/SP.J.1231.2013.38420

K. Lian, Li-li Zhao, Linlin Zhang, P. Jia, Lijie Tang, Junwei Ge, L. Yijing, Min Liu

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引用次数: 1

Abstract

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 IgG1 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∶ 105,1∶ 102 respectively.Their titers of ascites were 1∶ 108,1∶ 104 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.

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抗感染性胰腺坏死病毒VP2 COE蛋白单克隆抗体的制备及初步应用

重组蛋白VP2经Ni-NTA纯化后作为免疫原。用VP2蛋白腹腔免疫8周龄BALB/c小鼠3次，将免疫后的骨髓瘤细胞SP2/0与BALB/c小鼠脾细胞融合。经间接ELISA和极限稀释法筛选，获得2株抗VP2蛋白的杂交瘤细胞株，鉴定为IgG1亚型，命名为5G10、5F3。两种杂交瘤的染色体数在75 ~ 120之间。细胞培养上清抗体滴度分别为1∶105、1∶102。腹水滴度分别为1∶108、1∶104。Western-blot和间接免疫荧光分析表明，这两种单克隆抗体对IPNV具有特异性反应。间接ELISA结果显示，2个单克隆抗体对IHNV、VHSV、SVCV和HRV无反应能力。抗体加性实验表明，5G10和5F3识别IPNV VP2核蛋白的不同表位。我们检测了临床患IPN的虹鳟鱼肝脏组织材料，结果证实2个单克隆抗体可用于后续检测。

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来源期刊

水产学报 Environmental Science-Management, Monitoring, Policy and Law

CiteScore

1.40

自引率

0.00%

发文量

5213

期刊介绍： "Fisheries of" mainly reflects the results of scientific research and development of the direction of aquaculture for domestic and foreign academic exchanges Fisheries Service. Mainly basic research published in Fisheries, aquaculture and proliferation of fishing waters environmental protection, preservation of aquatic products processing and utilization, fishing equipment, and other aspects of mechanical papers, research briefings and reviewed.