禽流感病毒非结构蛋白1单克隆抗体的制备及其特异性分析

Preparation of monoclonal antibody against non-constructual protein-1 of avian influenza virus H5N1 and analysis of its specificity

为研制禽流感病毒(H5N1)非结构蛋白1(NS1)的特异性单克隆抗体(mAb),并鉴定其特异性,本研究在分别表达了具有良好抗原性的A/Vietnam/1194/04(H5N1)-NS1和A/HongKong/486/97(H5N1)-NS1重组蛋白基础上,用A/Viet-nam/1194/04(H5N1)-NS1蛋白免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞进行融合,间接ELISA筛选阳性的杂交瘤细胞,并结合免疫荧光和免疫印迹对抗体的特异性进行鉴定,通过竞争抑制实验对单抗识别的抗原位点进行分析。结果共获得19株能识别4个H5N1-NS1蛋白不同抗原位点的mAb,亚类测定显示,5株为IgG2a、1株为IgG2b,另外13株为IgG1。这些mAb均与A/Vietnam/1194/04(H5N1)-NS1和A/HongKong/486/97(H5N1)-NS1重组蛋白特异性结合,免疫荧光检测均与A型流感病毒(H1N1和H3N2)有交叉反应,而与B型流感病毒无交叉现象。表明成功获得特异性针对H5N1-NS1蛋白的mAb,为进一步研究禽流感病毒NS1蛋白的结构与功能奠定基础。

BALB/c mice were immunized with recombinant non-constructual protein-1（NS1）of A/Vietnam/1194/04（H5N1）and splenocytes of the immunized mice were fused with myeloma NS1 cells to produce hybridoma cell lines.ELISA assay was used to identify the monoclonal antibody against NS1 protein of H5N1 and immunofluorescence assay（IFA）and Western blotting were applied to identify the specificity of this antibody.It was demonstrated that 19 monoclonal antibodies recognizing 4 different epitopes on NS1 protein of H5N1 were obtained.These antibodies showed specific binding power to two recombinant NS1 protein of A/Vietnam/1194/94（H5N1）and A/Hongkong/486/07（H5N1）.The subtype of 5 of these antibodies was proved to be IgG2a,one of them was IgG2b and the other 13 were IgG1.As demonstrated by IFA,all of the antibodies strongly reacted with influenza A virus（H1N1 and H3N2）,yet without cross-reactivity with influenza B virus.It is evident that monoclonal antibodies against NS1 protein of H5N1 with high specificity had been successfully established,which may provide potential value for studies on the structural and functional characteristic of the NS1 protein of avian influenza virus H5N1.