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目的原核表达诺如病毒衣壳蛋白VP1,制备抗诺如病毒衣壳蛋白VP1的单克隆抗体,并研究其抗原表位特性。方法 PCR扩增GⅡ型湖州株诺如病毒VP1蛋白基因,将目的基因克隆至载体pET28a(+),转化至大肠埃希菌原核表达。纯化表达产物免疫BALB/c小鼠,将成功免疫的小鼠脾细胞与骨髓瘤SP2/0细胞融合。用Modeller9.9为诺如病毒GⅡ型湖州株VP1蛋白构建三维模型,构建好的三维模型提交到SEPPA在线B-cell空间表位预测软件,对GⅡ型湖州株VP1蛋白进行空间表位预测。用预测的表位序列来筛选阳性克隆,得到对应这些表位的单克隆抗体。结果成功构建重组表达载体pET28a(+)-Noro-VP1并在大肠埃希菌中获得表达,重组蛋白免疫的BALB/c小鼠的脾细胞与SP2/0细胞融合,用预测的抗原表位多肽筛选,获得10株杂交瘤细胞株。与重组蛋白的ELISA结果显示1E8、1P10、2C15和2L16四株反应性最强,其他几株反应性较弱。Western blotting结果显示只有2K10、1K16、1E8三株能与重组蛋白很好的结合。结论成功制备了抗GⅡ型湖州株诺如病毒VP1蛋白的单克隆抗体,抗原表位位于衣壳蛋白的P2区,为制备诺如病毒快速免疫诊断试剂盒及抗原表位的研究提供基础。
Objective To express Norovirus capsid protein VP1 in prokaryotic cells and prepare monoclonal antibody against Norovirus capsid protein VP1 and study its antigenic epitopes. Methods The VP1 gene of GV-type Norovirus was amplified by PCR. The target gene was cloned into vector pET28a (+) and transformed into E. coli for prokaryotic expression. BALB / c mice were immunized with the purified expression product, and successfully immunized mouse spleen cells were fused with myeloma SP2 / 0 cells. Modeller9.9 was used to construct three-dimensional model of norovirus VP2 protein of Huzhou strain GⅡ. The constructed three-dimensional model was submitted to SEPPA online B-cell space epitope prediction software to predict the spatial epitope of VP1 protein of GⅡ Huzhou strain. Positive clones were screened with the predicted epitope sequence to give the monoclonal antibodies corresponding to these epitopes. Results The recombinant expression vector pET28a (+) - Noro-VP1 was successfully constructed and expressed in Escherichia coli. The spleen cells of BALB / c mice immunized with the recombinant protein were fused with SP2 / 0 cells. The predicted epitope peptide Screening, access to 10 hybridoma cell lines. ELISA results with the recombinant protein showed that the four strains of 1E8, 1P10, 2C15 and 2L16 had the strongest reactivity and the other strains were less reactive. Western blotting showed that only 3 strains of 2K10, 1K16 and 1E8 could bind well with the recombinant protein. Conclusion The monoclonal antibody against VP1 protein of Norovirus of Huzhou strain was successfully prepared. The epitope was located in the P2 region of capsid protein, which provided the basis for the preparation of Norovirus rapid immunodiagnostic kit and antigen epitope.