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目的:建立能稳定分泌抗诺如病毒(Norovirus)N蛋白的单克隆抗体(mAb)细胞株,制备抗诺如病毒核衣壳蛋白的mAb,为诺如病毒的早期快速检测及致病机制的研究提供实验材料。方法:用E.coli表达的GⅡ组广州株NVgz01(DQ369797)Norovirus-N蛋白免疫BALB/c小鼠,通过PEG使小鼠脾细胞和Sp2/0细胞融合,使用HAT选择性培养基培养融合细胞,用间接ELISA和Western blot测定mAb的效价、免疫球蛋白亚型和mAb的特异性,并将各mAb之间进行配对。结果:通过细胞融合和克隆化,共筛选出4株分泌抗Norovir-us-N蛋白抗体的杂交瘤细胞株N2C3、N7C2、N4B1、N8A9。间接ELISA和Western blot检测结果表明,这4株mAb都可以与E.coli表达的GⅡ组广州株Norovirus-N蛋白产生特异性反应,并且能与天然粪便标本中的GⅡ组Norovirus产生特异性反应。配对结果显示N2C3和N7C2之间配对,对表达蛋白和天然病毒都具有较强的检测灵敏度。结论:获得了诺如病毒GⅡ组特异性mAb,为制备免疫诊断试剂盒及致病机制的研究奠定了基础。
OBJECTIVE: To establish a monoclonal antibody (mAb) cell line that can stably secrete anti-Norovirus N protein and prepare a mAb against Norovirus nucleocapsid protein, which is an early and rapid detection and pathogenic mechanism of Norovirus Research provides experimental materials. Methods: BALB / c mice were immunized with Norovirus-N protein of GⅡ group Guangzhou strain NVgz01 (DQ369797) by E.coli. The mouse spleen cells and Sp2 / 0 cells were fused by PEG, and the fusion cells were cultured using HAT selective medium , The mAb titers, immunoglobulin subtypes, and mAb specificity were determined by indirect ELISA and Western blotting, and the mAbs were paired. Results: Four hybridoma cell lines N2C3, N7C2, N4B1 and N8A9 secreting anti-Norovir-us-N antibody were screened by cell fusion and cloning. Indirect ELISA and Western blot results showed that all four mAbs reacted specifically with the Norovirus-N protein of Guangzhou strain of GⅡ group expressed in E. coli, and reacted specifically with Norovirus of GⅡ group in natural stool samples. Pairing results show that the pairings between N2C3 and N7C2 have strong detection sensitivity to the expressed protein and the natural virus. Conclusion: The specific mAb of Norovirus GⅡ group was obtained, which laid the foundation for the preparation of immunodiagnostic kit and the study of its pathogenesis.