流感病毒受体特异性红细胞检测试剂的制备及初步应用

Establishment of a protocol to label chicken red blood cells for identification of sialic acid receptor binding specificity of influenza virus

为优化流感病毒受体特异性的检测方法,本研究利用霍乱弧菌神经氨酸酶(VCNA)对鸡红细胞(RBC)进行去唾液酸化处理后,再分别通过α2,3和α2,6唾液酸转移酶以CMP-唾液酸作为底物进行不同唾液酸受体的标记,使其分别仅含SAα2,3Gal和SA2,6Gal受体,利用血凝试验和流式细胞仪检测其标记效果。流式检测结果显示,经过α2,3唾液酸转移酶处理的红细胞只含有SAα2,3唾液酸受体,而α2,6唾液酸转移酶只将SAα2,6唾液酸受体标记到红细胞表面。将两种红细胞分别经过人流感病毒和禽流感病毒的检测,表明该方法可以快速鉴定流感病毒的受体特异性。本研究所建立的流感病毒受体特异性标记和检测方法为流感病毒宿主嗜性范围的检测提供了一种有效的技术手段。

To develop a method to test the sialic acid （SA） receptor binding specificity of influenza virus, the chicken red blood cells （RBCs） were treated with vibrio cholera neuramindase （VCNA） to remove all the receptors on the surface of RBC, then re-labelled the SAα2,3Gal receptor or SAα2,6Gal receptor on the surface of the RBCs through two types of sialyltransferases. The normal RBCs, the VCNA treated RBC and the re-labelled RBCs were detected by flow cytometry with phytoagglutinin labeled by fluorescein isothiocyanate （FITC）, i.e. FITC-SNA and FITC-MAA. The SA receptor binding specificity of influenza viruses were identified by hemagglutination （HA） test with RBCs labeled with two differeut types of receptors. Flow cytometry showed that two types of RBCs only showed SAα2,3Gal or SA2,6Gal receptor, respectively. The result of testing on human and avian influenza virus strains showed that the re-labelled RBCs method could be used to rapidly and specifically identify the receptor binding specificity of influenza virus. The established protocol provided an alternative assay for rapidly detecting the host tropism of influenza virus.