禽流感病毒NP蛋白单克隆抗体的制备与鉴定

Preparation and Identification of Monoclonal Antibody against NP Protein of Avian Influenza Virus

为获得禽流感病毒(Avian influenza virus,AIV)NP蛋白的单克隆抗体,将构建的重组表达质粒pET-30a-NP转化BL21细胞,经IPTG诱导表达、纯化后作为免疫原,免疫8周龄Balb/c雌性小鼠,并按常规方法制备杂交瘤细胞。通过ELISA方法、Western-blot方法进行筛选,获得3株杂交瘤细胞株,命名为3A6、2H12、5F7,并进行了培养特性、分泌抗体活性、分泌抗体亚类的鉴定。结果显示:3株细胞株连续传10代均稳定分泌单克隆抗体,分泌的单克隆抗体亚型均为IgG2b,轻链类型均为Kappa。制备并纯化了以上3株单克隆抗体,浓度分别为2.9、2.5、2.8 mg/mL,纯度不低于90%。West-blot检测,单抗与H7血凝抑制试验抗原、H5血凝抑制试验抗原、H9N2病毒能发生特异性反应,说明单抗具有广谱性,且与IBDV、REV、IBV、MDV、ALV、AE、ILT、NDV、EDSV等均无特异性条带出现,说明特异性良好。本研究制备的3株针对禽流感NP蛋白的单抗,具有较好的特异性、保守性和广谱性,为下一步开展AIV诊断试剂如IFA检测试剂盒、ELISA检测试剂盒等的研制奠定了物质基础。

In order to obtain the monoclonal antibody against NP protein of Avian influenza virus(AIV),the recombinant expression plasmid pET30a-NP was transformed into BL21 cells.The recombinant plasmid pET30a-NP was induced by IPTG,purified and used as immunogen to immunize 8-week-old female Balb/c mice,and hybridoma cells were prepared by conventional methods.Three hybridoma cell lines,named 3A6,2H12 and 5F7,were obtained by ELISA,Western-blot and IFA method.Their culture characteristics,antibody secreting activity and subclass were identified.The results showed that the three cell lines stably secreted monoclonal antibodies for 10 passages,and the subtype of secreted monoclonal antibodies was IgG2b,light chain type was Kappa.The three strains of monoclonal antibody mouse ascites was prepared and purified,which concentration were 2.9,2.5,2.8 mg/mL separately,the purity were not less than 90%,The monoclonal antibody could react specifically with H7 hemagglutination inhibition test antigen,H5 hemagglutination inhibition test antigen,H9N2 virus,which indicated that the monoclonal antibody had broad spectrum.The monoclonal antibody had no specific bands with IBDV,REV,IBV,MDV,ALV,AE,ILT,NDV,EDSV,which indicated that the specificity was good.The three monoclonal antibodies against NP protein of AIV prepared in this study had good specificity,conservatism and broad-spectrum.This study layed a material foundation for the development of AIV diagnostic reagents such as IFA test kit,ELISA test kit in the next step.