抗H7N9亚型流感病毒神经氨酸酶单克隆抗体的制备及初步鉴定

摘要

目的 制备抗H7N9亚型流感病毒神经氨酸酶(Neuraminidase,NA)单克隆抗体,并对单抗的活性与功能进行初步鉴定.方法 利用免疫的BALB/c小鼠脾细胞与Sp2/0骨髓瘤细胞进行融合,获取杂交瘤细胞株,用间接ELISA方法筛选获得阳性杂交瘤细胞克隆,单克隆化后制备小鼠腹水单抗,纯化获得抗H7 N9亚型流感病毒NA的鼠源单克隆抗体,对抗体活性及功能进行鉴定.结果 获得特异性抗H7 N9亚型流感病毒N9鼠源单克隆抗体1G8、3C4和4E8,这三株抗体识别抗原表位不同,3C4和4E8能抑制神经氨酸酶活性,IC50分别为1.45 μg/ml和8.65 μg/ml.结论 3株单克隆抗体均特异性识别A/Anhui/1/2013(H7N9)的N9蛋白,有较好的N9结合活性或能抑制N9酶活性功能.本研究对新型H7N9禽流感病毒的诊断和防控提供了一个良好的思路.%Objective To develop the monoclonal antibody (mAb) against neuraminidase of H7N9 subtype influenza A virus and identify its biological function.Methods Female 8 week-old BALB/c mice were immunized and the splenocytes of the mice were fused with Sp2/0 myeloma cells.Indirect ELISA was used to screen hybridoma and the positive clones were subject to be subcloned.Positive clones were identified and the monoclonal antibodies(mAbs) were obtained by purifying the ascetic fluid of mice injected with the hybridoma.The NA-binding as well as neuraminidase-inhibition activity of these mAbs were determined.Results Three mAbs against neuraminidase of H7N9 subtype influenza A virus,1G8,3C4 and 4E8,were obtained.Thcy dcmonstrated different epitop-recognizing.3C4 and 4E8 exhibited neuraminidase inhibitory activity,with a IC50 of 1.45 μg/ml and 8.65 μg/ml,respectively.Conclusions The results suggested that mAbs specific to neuraminidase of H7N9 subtype influenza A virus were developed,providing an useful tool in control and preventing the novel H7N9 influenza A virus.