GⅡ型诺如病毒衣壳蛋白抗原表位预测及单克隆抗体的制备

陈莉萍; 纪蕾; 吴晓芳; 徐德顺; 韩建康

doi:10.3784/j.issn.1003-9961.2014.01.018

GⅡ型诺如病毒衣壳蛋白抗原表位预测及单克隆抗体的制备

doi: 10.3784/j.issn.1003-9961.2014.01.018

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出版历程
- 收稿日期: 2013-01-10
- 刊出日期: 2014-01-20

Capsid protein antigen epitope prediction and monoclonal antibody preparation for Norovirus GⅡ

摘要

摘要: 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。
- 诺如病毒 /
- 衣壳蛋白 /
- 单克隆抗体 /
- 抗原表位
Abstract: Objective To express the noruvirus capsid protein(VP1), prepare monoclonal antibodies against it and understand the noruvirus capsid proteins epitope characteristics. Methods PCR amplification of VP1 gene of Norovirus GⅡ(Huzhou strains ) was conducted, the clone of target gene to the vector pET28a(+) and transferring into E. coli were conducted too. BALB/c mice were immunized with purified expression product. The spleen cells of successfully immunized mice were fused with myeloma cells SP2/0. Protein modeling was performed with program Modeller 9.9. The modeled 3D structures of capsid protein of norovirus were submitted to the SEPPA for predicting the possible spatial epitopes with the default threshold. Positive clones were screened to obtain monoclonal antibodies corresponding to these epitopes by predicted epitope sequence. Results The recombinant expression vector pET28a(+)-Noro-VP1 was successfully constructed and expressed in E. coli, the recombinant protein-immunized BALB/c mouse spleen cells were fused with SP2/0 cell. The predicted epitope region polypeptide screened ten Hybridoma. Indirect ELISA assay showed that 1E8, 1P10, 2C15 and 2L16 can react with recombinant protein strongly, the other 6 mAbs had weak reaction. Western blotting showed that only 2K10, 1K16, 1E8 can specifically recognized the Norovirus capsid protein expressed in E.coli. Conclusion The mAbs against G Ⅱ norovirus capsid protein was successfully prepared. Epitopes was in capsid protein P2 region. This can be used for the development of rapid immunodiagnostic kits and epitopes research of norovirus capside protein.
- norovirus /
- capsid protein /
- monoclonal antibody /
- epitopes

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参考文献(16)

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文章访问数: 1574
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GⅡ型诺如病毒衣壳蛋白抗原表位预测及单克隆抗体的制备

doi: 10.3784/j.issn.1003-9961.2014.01.018

计量

出版历程

Capsid protein antigen epitope prediction and monoclonal antibody preparation for Norovirus GⅡ

计量

出版历程

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