3型鲤疱疹病毒白介素10单克隆抗体制备与鉴定

Preparation and characterization of Cyprinid herpesvirus 3 interleukin 10 monoclonal antibody

白介素(Interleukin,IL)10是脊椎动物Th2细胞(T helper 2 cell)和先天淋巴细胞(Innate lymphoid cell)分泌的一种多效应细胞因子。3型鲤疱疹病毒(Cyprinid herpesvirus 3,CyHV3)ORF134编码IL10样蛋白,本研究构建了ORF134原核表达质粒,在大肠杆菌DE3感受态细胞中表达CyHV3-IL10重组蛋白。SDSPAGE分析结果表明,重组CyHV3-IL10蛋白约为18 ku,主要以包涵体形式表达。将CyHV3-IL10包涵体进行变性和复性,经分子筛凝胶柱层析纯化获得了纯度较高的重组CyHV3-IL10蛋白,利用CyHV3-IL10重组蛋白免疫小鼠制备了单克隆抗体,通过酶联免疫吸附测定法(Enzyme linked immunosorbent assay,ELISA)筛选,获得了2株亲和力强的抗体,并利用激光共聚焦技术和Western blotting对抗体进行鉴定。结果表明,FITC标记的单克隆抗体能够特异性识别HEK293细胞中表达的CyHV3-IL10重组蛋白和感染CyHV3的镜鲤脾和鳃组织中表达的CyHV3-IL10蛋白。CyHV3-IL10单克隆抗体的制备为后续研究CyHV3-IL10蛋白功能和建立CyHV3诊断方法奠定基础,为CyHV3病毒检测试剂盒的研发提供新思路。

Cyprinid herpesvirus 3(CyHV3),also known as Koi herpesvirus(KHV),is a highly contagious pathogen that threatens the sustainability of cyprinid aquaculture.The ORF134 gene of CyHV3 encodes an interleukin(IL)10 like protein.In this study,we constructed a prokaryotic expression plasmid of ORF134 and transformed it into the E.coli Rosetta(DE3)cells for expression of recombinant protein.The recombinant CyHV3-IL10 protein was purified by size exclusion chromatography and used for immunization of mice to generate monoclonal antibodies.The antibodies were then characterized by ELISA,Western blotting and immunofluorescent assay.SDS-PAGE analysis showed that the recombinant CyHV3-IL10 protein was approximately 18 ku in size and was expressed mainly in the form of inclusion bodies.The inclusion bodies were denatured,refolded and subjected to size exclusion chromatography.The recombinant CyHV3-IL10 protein was of high purity.Western blotting and immunofluorescence analyses revealed that the CyHV3-IL10 monoclonal antibodies recognized the recombinant CyHV3-IL10 protein expressed in the E.coli and HEK293 cells and could specifically detect the HEK293 cells expressing the CyHV3-IL10 protein.In summary,the availability of CyHV3-IL10 monoclonal antibodies lays the foundation for the functional study of CyHV3-IL10 and the establishment of CyHV3 diagnostic tools,and provides novel strategies for the prevention and control of the diseases caused by CyHV3.