猪流感病毒NP蛋白单克隆抗体的制备与鉴定

Preparation and Identification of Monoclonal Antibodies against NP Protein of Swine Influenza Virus

为了制备猪流感病毒(SIV)核蛋白(NP)的单克隆抗体(MAb),采用差速离心法纯化H1N1和H3N2亚型SIV后交叉免疫BALB/c小鼠,利用淋巴细胞杂交瘤技术制备杂交瘤细胞,通过建立的免疫过氧化物酶单层细胞试验(IPMA)单克隆抗体检测方法进行筛选。获得3株能稳定分泌抗NP蛋白的杂交瘤细胞,分别命名为16D5、18G8和20C4,将它们诱导小鼠产生的腹水IPMA效价分别为1×10^-6、1×10^-6和1×10^-5。亚型鉴定结果显示,3株单克隆抗体的重链均为IgG1,轻链为κ链。3株单克隆抗体特异识别H1N1、H3N2、H5N1、H7N9和H9N2亚型流感病毒,并且与猪圆环病毒2型、猪伪狂犬病毒和猪细小病毒无交叉反应。Westernblot检测结果表明,3株杂交瘤细胞培养上清均在56ku附近出现一条特异性的蛋白质条带,说明针对SIV的NP蛋白制备了3株单克隆抗体。综上,成功制备了针对SIVNP蛋白的3株单克隆抗体,可识别不同亚型的SIV。

In order to prepare monoclonal antibodies(MAb) against swine influenza virus(SIV),BALB/c mice were cross-immunized with the purified H1N1 and H3N2 subtype SIV,lymphocyte hybridoma technology was used to prepare hybridoma cells, and the positive hybridoma cells were screened by the established immunoperoxidase monolayer assay(IPMA) monoclonal antibody detection method.The hybridoma cells secreting anti-NP protein were obtained and named 16D5,18G8 and 20C4,respectively.The IPMA titers of ascites produced by monoclonal antibodies in mice were 1×10^-6 , 1×10^-6 and 1×10^-5.The subtype identification results showed that the heavy chains of the three monoclonal antibodies were all IgG1 and the light chain was κ chain.The three monoclonal antibodies recognized the H1N1,H3N2,H5N1,H7N9 and H9N2 subtype influenza viruses specifically,and porcine circovirus type 2,pseudorabies virus and porcine parvovirus did not cross-react.Western blot assay showed that the three hybridoma cells produced a specific band around 56 ku,indicating that three monoclonal antibodies were prepared against SIV NP protein.In summary,three monoclonal antibodies against SIV NP protein were successfully prepared to recognize different subtypes of SIV.