猪塞尼卡谷病毒单克隆抗体的制备及鉴定

Preparation and Identification of Monoclonal Antibodies Against Porcine Seneca Valley Virus

【目的】纯化猪塞尼卡谷病毒(Seneca Valley virus,SVV)SVV-CH-HB2016毒株,并制备其结构蛋白VP1、VP2和VP3的单克隆抗体。【方法】以蔗糖密度梯度离心法纯化的SVV-CH-HB2016病毒颗粒作为抗原,免疫BALB/c小鼠,取脾细胞与骨髓瘤细胞(SP2/0)进行细胞融合。通过间接免疫荧光试验(IFA)结合间接ELISA筛选阳性细胞株,制备能特异性分泌针对结构蛋白的杂交瘤细胞株。采用Western blotting和IFA方法分别检测单克隆抗体与重组表达蛋白及天然结构蛋白的反应性,并对单克隆抗体的病毒中和保护效果进行测定。利用空斑试验和实时荧光定量PCR方法探究中和性单克隆抗体对SVV-CH-HB2016毒株吸附过程的影响,最后用抗体相加试验来分析14株单克隆抗体的抗原表位。【结果】在蔗糖密度梯度为5%~45%(W/V)时获得了纯度较好、浓度较高的SVV-CH-HB2016毒株结构蛋白,免疫小鼠血清抗体效价均达到了1∶12800,成功制备了17株能稳定分泌特异性单克隆抗体的杂交瘤细胞株。经验证14株单克隆抗体能与重组结构蛋白发生Western blotting反应,17株单克隆抗体能与病毒发生IFA作用;2G6、4A3和4C113株单克隆抗体对SVV-CH-HB2016毒株感染的BHK-21细胞具有明显中和保护作用,也能有效抑制SVV-CH-HB2016毒株对293T细胞的吸附。经分析发现,除1F5与2E1、4B8与4F11外,其余10株单克隆抗体分别针对不同抗原表位。【结论】本研究初步建立了SVV的纯化方法,制备了17株特异性针对SVV-CH-HB2016毒株的单克隆抗体,为后期进一步开展SVV全病毒灭活疫苗的研发、ELISA检测方法的建立及保护性抗原表位的鉴定奠定了基础。

【Objective】The aim of this study was to purify SVV-CH-HB2016 strain and prepare monoclonal antibodies of Seneca Valley virus(SVV)specific to the structural proteins VP1,VP2 and VP3.【Method】SVV-CH-HB2016 virus concentrate was purified using sucrose density gradient centrifugation and used as antigen to immunize BALB/c mice.Immune spleen cells and myeloma cells(SP2/0)were used in cell fusion experiment.Both indirect immunofluorescence assay(IFA)and indirect ELISA were conducted to screen positive cell lines which could secrete hybridoma cell lines specific to structural proteins.The reactivity of monoclonal antibody with recombinant and natural structural proteins was tested using Western blotting and IFA,the neutralization and adsorption effects of monoclonal antibody were also tested.Then plaque assay and Real-time PCR were used to explore the effect of neutralized monoclonal antibody on the absorption process of SVV-CH-HB2016 strain.Finally,the antibody addition test was used to analyze the epitope of the monoclonal antibody.【Result】Both the purity and concentration of SVV-CH-HB2016 strain structure protein could keep a higher level when sucrose gradient was between 5%-45%(W/V).The serum antibody titer of the immunized mice reached 1∶12800,and successfully prepared 17 hybridoma cell lines capable of stably secreting specific monoclonal antibody.It was found that 14 monoclonal antibody strains could react with recombinant structural proteins in Western blotting,and 17 monoclonal antibody strains could react with the virus in IFA.Strains 2G6,4A3 and 4C11 not only had a significant neutralizing and protective effect on BHK-21 cells infected by SVV-CH-HB2016 strain but also could effectively inhibit the adsorption of SVV-CH-HB2016 strain on 293T cells.It was found that in addition to 1F5 and 2E1,4B8 and 4F11,the other 10 monoclonal antibody strains were specific to different epitopes.【Conclusion】In this study,the purification method of SVV was preliminarily established,and 17 monoclonal antibody strains specific to SVV-CH-HB2016 strain were prepared,which laid a foundation for the further development of inactivated SVV vaccine,the establishment of ELISA detection method and the identification of protective antigen epitope in the future.