SARS-CoV S666蛋白与眼部ACE2受体的结合作用

Binding action between SARS-CoV S666 protein and ACE2 receptor in eyes

目的研究SARS冠状病毒(severe acute respiratory syndrome coronavirus,SARS-CoV)S666蛋白与眼部功能性受体-血管紧张素转化酶2(angiotensin-converting enzyme2,ACE2)的结合作用。方法构建SARS-CoV S666蛋白融合基因pS666-EGFP,并在哺乳动物细胞(293T细胞)中表达;酶联免疫吸附实验及流式细胞仪检测法进行SARS-CoV S666蛋白与结膜、角膜细胞及心、肺组织的体外结合研究。结果构建了融合基因pS666-EG-FP,琼脂糖凝胶电泳、SDS-PAGE及Western Blot分析证明SARS-CoV pS666-EGFP融合基因构建正确,基因表达产物与理论推算值相符,相对分子量约为100.7×103。酶联免疫吸附实验结果经析因方差分析显示:S666-EGFP蛋白与角膜上皮细胞、结膜成纤维细胞、结膜上皮细胞及结膜、角膜组织的结合能力比EGFP蛋白强(有统计学差异,P<0·001)。流式细胞仪检测结果显示:结膜上皮细胞、结膜成纤维细胞、角膜上皮细胞与S666-EGFP蛋白结合,但与EGFP蛋白均无结合。上述细胞及组织的结合能力较vero E6细胞及心、肺组织的结合能力稍弱。结合反应能够被ACE2抗体所阻滞,证实其为特异性结合。结论SARS-CoV S666蛋白与结膜、角膜细胞能够结合,对了解SARS的病理机制及进一步研究SARS的防护和治疗具有一定的参考意义。

Objective To investigate the binding action between the severe acute respiratory syndrome coronavirus （SARS-CoV） S666 protein and the angiotensin-converting enzyme 2 （ ACE2 ） receptor in eyes. Methods The recombinant pS666-EGFP were transfected into 293T ceils and expressed, the binding capability of the SARS-CoV S666 proteins with ceils including vero E6 ceils, conjunctival epithelial ceils, conjunctival flbroblast and corneal epithelial ceils and tissues including heart, lung, biain, cornea and conjunctiva were examined by enzyme-linked immunosorbent assay and flow cytometry. Results The map of restriction enzyme digestion showed the pS666-EGFP fusion gene was successfully cloned into the vector pEGFP-N1. Western Blotting showed that the relative molecular weight of fusion protein was about 100.7× 10^3 after transfection of 293T ceils with pS666-EGFP. The enzyme-linked immunosorbent assay showed, compared with the control group of EGFP proteins, the binding level of S666- EGFP proteins increased in epithelial ceils, conjunctival fibroblast, corneal epithelial ceils,corneal and conjunctival tissue （P 〈0.001 ） ,but the binding capabilities of the above ceils and tissues were weaker than that of veto E6 ceils ,heart and lung tissue. The results of flow cytometry fitrther confirmed this results, and this interaction could be blocked by a goat anti-ACE2 antibody,which indicated that the interaction was specific. Conclusion The SARS-CoV S666 protein can bind with the conjunctival and corneal ceils slightly, which is helpful not only for understanding the mechanism of SARS,but for the further study on the prevention and treatment of SARS.