H7N9亚型流感病毒特异性中和抗体的筛选及其表位的鉴定

Screening and epitope identification of specific neutralizing antibodies against H7N9 subtype influenza virus

为了获得H7N9亚型流感病毒的中和抗体以及鉴定新的中和表位,以原核表达的H7N9亚型流感病毒SZ19毒株血凝素蛋白HA1亚基作为免疫原,通过杂交瘤细胞技术,构建筛选得到4株单克隆抗体杂交瘤细胞株2B2B3、3C5D6、4E12D6、9F10G3;将杂交瘤细胞扩大培养后,注射免疫6周龄雌性BALB/c小鼠腹腔,制备获得4株单克隆抗体;对制备的4株单克隆抗体的特异性、ELISA效价、中和活性等生物活性进行鉴定与筛选。结果表明,筛选获得1株能够特异性识别H7N9亚型流感病毒HA蛋白的中和单克隆抗体2B2B3,其半数抑制浓度(IC_(50))为67.8 ng/μL,ELISA效价为4 096,为具有κ轻链的Ig G2b亚类,并通过肽扫描技术鉴定出H7N9亚型流感病毒HA蛋白高度保守的中和表位^(115)ALRQILRESGGIDKEA^(130)。

In order to obtain neutralizing antibodies against H7N9 influenza virus and identify new neutralizing epitopes,the prokaryotic hemagglutinin (HA1) protein of SZ19 strain of H7N9 influenza virus was used as immunogen in this study.Four monoclonal antibody hybridoma cell lines 2B2B3,3C5D6,4E12D6and 9F10G3 were constructed and screened.Hybridoma cells were cultured and injected into the abdominal cavity of 6-week-old female BALB/c mice to prepare four strains of monoclonal antibodies.The specificity,ELISA titer,neutralization activity and other biological activities of the four monoclonal antibodies were identified and screened.The results showed that a neutralizing monoclonal antibody 2B2B3 that could specifically recognize the HA protein of H7N9 influenza virus was obtained.Its IC_(50) was67.8 ng/μL,ELISA titer was 4 096,and it was an Ig G2b subclass withκlight chain.The highly conserved neutralizing epitope ^(115)ALRQILRESGGIDKEA^(130) of HA protein of H7N9 influenza virus was identified by peptide scanning.