| 摘要: |
| [目的]制备猪繁殖与呼吸综合征病毒(PRRSV)的单克隆抗体,探讨单克隆抗体临床应用的价值及病毒蛋白的结构和功能。 [方法] 采用差速和蔗糖不连续密度梯度离心纯化的PRRSV抗原免疫Balb/c小鼠,取免疫小鼠的脾细胞与SP2/0骨髓瘤细胞常规融合, 经间接ELISA的筛选和有限稀释亚克隆,获得能稳定分泌鼠抗PRRSV单克隆抗体的杂交瘤细胞株,并通过ELISA、Western blot和免疫荧光试验等方法分析其特性。[结果] 成功地获得了3株稳定分泌抗PRRSV-GP5蛋白的单克隆抗体杂交瘤细胞株,分别命名为A61、B47和C65。这3株单克隆抗体的免疫球蛋白亚类分别为IgG1、IgG3和IgG1,均具有ELISA、IFA和Western blot特性,细胞上清液和腹水的ELISA抗体效价分别为1×27～1×28和1×105～1×106。特异性测定结果表明,3株单克隆抗体与Marc-145细胞、猪2型圆环病毒和猪细小病毒均无交叉反应。Western blot 测定结果表明,3株单克隆抗体均能特异性识别PRRSV-GP5蛋白。[结论] 获得了特异性识别PRRSV-GP5蛋白的单克隆抗体,为PRRS免疫诊断试剂盒的研制奠定了基础。 |
| 关键词: 猪繁殖与呼吸综合征病毒 单克隆抗体 酶联免疫吸附试验 免疫荧光试验 Western blot |
| DOI： |
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| 基金项目:福建省重大专项（2006NZ0003-2） |
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| Production and Characterization of Monoclonal Antibodies against Porcine reproductive and respiratory syndrome virus |
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| Abstract: |
| 【Objective】 The paper was to obtain monoclonal antibodies against PRRSV for further study of its structure and function.【Method】 The antigen of PRRSV FZ strain was purified by differential centrifugation and discontinuous sucrose density gradient centrifugation.The spleen cells of Balb/c mice immunized with purified viral antigen were fused with SP2/0 myeloma cells.Hybridoma cell lines secreting monoclonal antibodies against PRRSV were screened by using indirect enzyme linked immunosorbent assay(ELISA) and the subcloning approach.The specificities of these monoclonal antibodies were determined by ELISA,Western blotting and Immunofluorescecence assay.【Result】 Three hybridoma cell lines developed could steadily secure specific monoclonal antibodies(McAbs) against PRRSV-FZ and were designated A61,B47 and C65,respectively.The isotyping analysis results showed that both A61 and C65 belonged to IgG1,and B47 belonged to IgG3.These McAbs could bind to the antigen of PRRSV FZ in ELISA,IFA and western-blot analysis.Their ELISA titers of the supernatant ranged from 27 to 28 and that of ascites fluid was 105-106.These McAbs were highly specific and had no cross-reactivity with Marc-145 cells，Porcine parvovirus(PPV) and Porcine circovirus Type 2 (PCV2).Western blot analysis confirmed that three McAbs could recognize 26 Ku structure protein(GP5 protein).【Conclusion】 3 Monoclonal antibodies of high specificity against GP5 protein were prepared successfully,which laid a foundation for the further study on PRRSV quick-diagnosis. |
| Key words: PRRSV monoclonal antibody ELISA IFA Western blot |
