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目的寻找HeLa细胞膜上可与日本脑炎病毒(JEV)结合的分子。方法采用免疫共沉淀(Co-IP)技术,分离HeLa细胞膜上可与JEV特异结合的分子,对SDS-PAGE特异条带进行质谱分析,并经Western blotting和酶联免疫吸附试验(ELISA)验证其与JEV的特异结合。结果 Co-IP复合物经SDS-PAGE分离出多个条带,选择最明显的3条带进行了质谱分析,结果显示分别为真核细胞翻译延长因子2(eEF2)、热休克蛋白90β(HSP90β)和微管蛋白α6。利用兔抗人HSP90β单克隆抗体进行Western blotting和ELISA实验,证实了细胞膜中确有HSP90β且可与JEV特异性结合。结论 HSP90β是HeLa细胞膜JEV受体的候选分子。
Objective To find a molecule that can bind to Japanese encephalitis virus (JEV) in HeLa cell membrane. Methods Specific co-immunoprecipitation (Co-IP) technique was used to separate the specific binding molecules of JEV on HeLa cell membrane. The specific bands of SDS-PAGE were analyzed by mass spectrometry and Western blotting and enzyme-linked immunosorbent assay (ELISA) Specific binding to JEV. Results The bands of Co-IP complex were separated by SDS-PAGE, and the most obvious three bands were selected for mass spectrometry. The results showed that eEF2, HSP90β ) And tubulin α6. Western blotting and ELISA assay using rabbit anti-human HSP90β monoclonal antibody confirmed that there was indeed HSP90β in the cell membrane and specific binding to JEV. Conclusion HSP90β is a candidate molecule of JEV receptor in HeLa cell membrane.