抗H9亚型禽流感病毒血凝素单克隆抗体的制备和鉴定

目的:获得分泌抗H9亚型禽流感病毒(AIV)血凝素单克隆抗体的杂交瘤细胞。方法:以H9N2亚型AIV为免疫原,免疫6～8周龄雌性BALB/c小鼠,取其脾细胞与骨髓瘤细胞Sp2/0-Ag-14,在PEG4000的作用下进行细胞融合,通过血凝抑制(HI)试验筛选分泌抗H9亚型AIV血凝素单克隆抗体的杂交瘤细胞。结果:经过连续3～4次克隆化,获得能稳定分泌抗H9亚型AIV血凝素的单克隆抗体细胞系6株,分别命名为1B2、1C10、1G2、2B7、2E3和5E11。6株细胞培养上清HI效价为24～28,腹水HI效价为210～213。除1G2为IgM外,其余5株均为IgG1。Western blotting结果显示,1B2、1C10、2B7和2E3能与AIVH9蛋白在Mr为75000处反应,表明其是针对AIVH9亚型血凝素蛋白的单抗。特异性试验表明该6株单抗均只与H9亚型AIV发生特异性HI反应,而不与其他14个HA亚型的AIV及新城疫病毒、传染性支气管炎病毒发生交叉反应,显示出良好的特异性。结论:制备了针对H9亚型禽流感病毒血凝素的单克隆抗体,为禽流感的快速诊断和病毒的抗原性分析等奠定了基础。

Development and Characterization of Monoclonal Antibodies Against Hemagglutinin of H9 Subtype Avian Influenza Virus

Objective：To develop and characterize monoclonal antibodies（McAbs）against hemagglutinin of H9 subtype avian influenza virus（AIV）.Methods：Six to eight-week-old female mice were immunized with H9 subtype AIV.Splenocytes from the immunized mice were fused with Sp2/0-Ag-14 myeloma cells.Hemagglutinin inhibition （HI）test was used to screen hybridoma cells and limited dilution method was performed to subclone the positive clones.Results：After 3～4 cycles of subcloning,six hybridoma cell strains secreting specific McAb were established and designated as 1B2,1C10,1G2,2B7,2E3 and 5E11,respectively.The HI titers in their supernatants were 24～28,and those in ascites were 210～213.Except the immunoglobulin subclass of 1G2 was IgM,all other McAbs were belonged to IgG1.Western blotting analysis confirmed that the McAbs of 1B2,1C10,2B7 and 2E3 only reacted with Mr 75 000 HA protein of H9 subtype AIV.The results of reactivity showed that all the six obtained McAbs only reacted with H9 subtype of AIV but did not react with other subtypes of AIV,Newcastle disease virus and infectious bronchitis virus in HI assay.Conclusion：The results suggested that McAbs specific to hemagglutinin of H9 subtype AIV were developed.These McAbs would be a useful tool for AIV surveillanc,rapid clinical diagnosis, and antigenic epitope analysis.