诺如病毒表达衣壳蛋白单克隆抗体的制备、鉴定和初步应用

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Norovirus-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.

Preparation, characterization and preliminary application of monoclonal antibodies against the expressed Norovirus capsid protein

AIM: To prepare the monoclonal antibodies (mAbs) against Norovirus capsid protein for the development of a rapid assay of Norovirus and to investigate the pathogenesis of this virus. METHODS: Sp2/0-Ag-14 myeloma cells were fused with spleen cells of BALB/c mice immunized with the recombinant protein of Norovirus NVgz01 (DQ369797), which was overexpressed in E.coli. The monoclonal antibodies against Norovirus capsid protein were screened by selective culture medium. The obtained Ig subtypes, titer and specificity of the mAbs were examined by ELISA and Western blot respectively. RESULTS: After cell fusion and subcloning, four hybridoma cell lines which secreted monoclonal antibodies specifically against Norovirus capsid protein were obtained and named as N2C3, N7C2, N4B1 and N8A9. Indirect ELISA and Western blot assay showed that the four mAbs specifically recognized the Norovirus capsid protein expressed in E.coli. The native Norovirus capsid protein in the stool samples were also recognized by them. The Sandwich ELISA, a rapid detection assay of the expressed and native Norovirus capsid protein, demonstrated that N2C3 and N7C2 were matched successfully. CONCLUSION: The mAbs against GII Norovirus capsid protein have been developed, which can be used for the development of detection assay and the basic research of Norovirus.