Development and identification of monoclonal antibody against recombinant major capsid protein of infectious spleen and kidney necrosis virus from Siniperca chuatsi
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摘要: 为了建立鳜传染性脾肾坏死病毒(ISKNV)疫苗抗原含量的ELISA检测方法,制备了3株抗ISKNV主衣壳蛋白(MCP)的单克隆抗体,鉴定了其生物学特性。将大肠杆菌表达的重组MCP纯化复性后,连续3次免疫BALB/c小鼠,然后将免疫小鼠的脾细胞与SP2/0细胞融合,经过克隆、筛选,获得3株能稳定分泌抗ISKNV MCP蛋白的单克隆抗体阳性细胞株,分别命名为5F1、3D9和5B4,均为IgG1亚型。间接ELISA实验表明,3株单抗可特异性识别ISKNV,与鳜弹状病毒、大鲵虹彩病毒等无交叉反应。将5F1株免疫小鼠后制备腹水,以重组MCP和ISKNV细胞培养物上清液为检测抗原,ELISA检测腹水效价分别为1:51 200和1:400。间接免疫荧光(IFA)和Western Blotting鉴定结果显示,5F1能够与ISKNV病毒发生特异性反应,并初步确定5F1单抗株制备的腹水用于IFA的使用浓度为1:200、Western Blotting的使用浓度为1:1000。结果证实,成功制备了抗ISKNV MCP的单克隆抗体,可特异性识别ISKNV病毒粒子和MCP蛋白,为建立ISKNV疫苗抗原含量检测方法奠定了基础。Abstract: Infectious spleen and kidney necrosis virus (ISKNV) causes a disease with high mortality, resulting in significant economic loss to Siniperca chuatsi culture industry in China. To establish ELISA assay to determine virus antigen content of ISKNV vaccine and investigate the pathogenic mechanism of ISKNV major capsid protein (MCP), the monoclonal antibody (MAb) against recombinant MCP protein was developed and the properties were identified. The purified recombinant MCP was injected into BALB/c mice through subcutaneous route for three times. Then myeloma cells SP2/0 were fused with the spleen cells of the immunized BALB/c mice. Three hybridoma cell lines against ISKNV MCP were screened using indirect ELISA and were identified to be IgG1 subtype, designated as 5F1, 3D9 and 5B4, respectively. The three McAbs had no reaction with SCRV and STIV except ISKNV by the indirect ELISA. The hybridoma cell line 5F1 was selected for ascites preparation. When the recombinant MCP and ISKNV supernatant was used as detective antigen respectively, the titer of ascites was 1:51 200 and 1:400 respectively. Indirect immunofluorescence and Western-blot analysis showed that the McAb 5F1 could recognize authentic MCP protein of ISKNV and working concentration of McAb 5F1 was confirmed. From above, the MAb against MCP of ISKNV was successfully prepared, which laid a foundation for further study.
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图 ISKNV重组MCP蛋白的SDS-PAGE分析 1. 复性的重组MCP蛋白；2. 纯化的重组MCP蛋白；3. 诱导4 h的重组菌(DH5α/ pBVMcp)；4. 诱导4 h的培养基上清液；M. 蛋白质分子质量标准蛋白
Figure . SDS-PAGE analysis of the recombinant MCP protein of ISKNV 1. renatured recombinant MCP protein; 2. purified recombinant MCP protein; 3. recombinant DH5α (pBV220) 4 h post inducement; 4. culture supernatant; M. protein molecular weight marker
图 3株杂交瘤细胞上清液与感染ISKNV的CPB细胞的间接免疫荧光染色结果 (a) 3D9; (b) 5B4; (c) 5F1
Figure . IFA result of CPB cells inoculated with ISKNV recognized by supernant of 3 hybridoma cell strains
图 不同稀释度5F1腹水与感染ISKNV的CPB细胞的间接免疫荧光结果 (a) 1∶200稀释，明场；(b) 1∶200稀释，荧光；(c) 1∶500稀释，明场；(d) 1∶500稀释，荧光；(e) 1∶1000稀释，明场；(f) 1∶1000稀释，荧光
Figure . IFA result of CPB cells inoculated with ISKNV recognized by 5F1 ascites at different dilution (a) at a dilution of 1: 200, phase contrast; (b) at a dilution of 1: 200, FITC; (c) at a dilution of 1: 500, phase contrast; (d) at a dilution of 1: 500, FITC; (e) at a dilution of 1: 1000, phase contrast；(f) at a dilution of 1: 1000, FITC
图 不同稀释度5F1腹水与ISKNV免疫印迹结果 1. ISKNV病毒培养液浓缩50倍，1∶1000稀释的5F1腹水；2. ISKNV病毒培养上清液，1∶500稀释的5F1腹水；3. ISKNV病毒培养上清液，1∶1000的5F1腹水；M．预染蛋白marker
Figure . Western-blotting identification of 5F1 ascites reacting with ISKNV at different dilution 1. ISKNV concentrated by 50 times, ascites at a dilution of 1∶1000; 2. ISKNV supernant, ascites at a dilution of 1∶500; 3. ISKNV supernant, ascites at a dilution of 1∶1000; M. protein marker
表 抗ISKNV重组MCP单克隆抗体亚型鉴定(OD490)
Table . The subtype identification of monoclonal antibody against ISKNV (OD490)
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