广谱性识别H7亚型禽流感病毒HA蛋白的单克隆抗体制备与鉴定

Preparation and Identification of Monoclonal Antibody Broadly Recognizing HA Protein of H7 Subtype Avian Influenza Virus

H7N9亚型禽流感病毒(AIV)的快速进化与变异使得其逃脱现有抗体的识别,为提供H7亚型AIV鉴别诊断所需重要材料,制备可识别H7亚型AIV HA蛋白的单克隆抗体(mAb)。采用差速离心法纯化的H7N9亚型AIV(A/Chicken/Guangdong/SW154/2015)免疫BALB/c小鼠,利用杂交瘤技术制备杂交瘤细胞,采用血凝抑制(HI)试验、免疫过氧化物酶单层细胞试验(IPMA)和间接ELISA检测方法筛选阳性杂交瘤细胞株。结果显示,获得5株稳定分泌抗H7N9 AIV HA蛋白mAb的杂交瘤细胞,腹水ELISA效价均为1∶1000000;其中,4株mAb的重链为IgG1,1株mAb的重链为IgG2b,轻链均为κ链。特异性测定结果显示,5株mAb仅与H7亚型AIV反应,不与其他亚型流感病毒发生反应;广谱性测定结果显示,5株mAb均可与不同年份分离出的H7亚型AIV发生反应;HI结果显示,腹水针对H7-Re3抗原株的HI效价为7 log2~12 log2;MDCK细胞微量中和试验结果显示,5株mAb均具有中和活性;Dot blot检测结果显示,5株mAb均可识别H7-Re2抗原株和H7-Re3抗原株;Western blot检测结果表明,5株杂交瘤细胞培养上清均在63 ku附近出现1条特异性的蛋白质条带,说明这5株mAb均识别线性表位。综上,成功制备了5株广谱性识别H7 AIV HA蛋白的单克隆抗体。

The rapid evolution and mutation of H7N9 subtype avian influenza virus(AIV)made it escape the recognition of existing antibodies.Monoclonal antibodies(mAbs)were prepared that broadly recognized the HA protein of H7 subtype AIV,which would provide effective antibodies for the diagnosis of H7 subtype AIV.BALB/c mice were immunized with H7N9 subtype AIV(A/Chicken/Guangdong/SW154/2015)purified by differential centrifugation,and hybridoma cells were prepared by hybridoma technology.MAbs were screened by hemagglutination inhibition assay(HI),immunoperoxidase monolayer assay(IPMA)and indirect ELISA.Five strains of hybridoma cells that stably secreted mAbs anti-HA protein were obtained,and the ELISA titers of ascites were all 1∶1000000.Among them,the heavy chain of four mAbs was IgG1,the heavy chain of one mAb was IgG2b,and the light chains were allκchain.The specificity assay showed that the five mAbs only reacted with H7 subtype AIV and did not react with other subtype of influenza viruses.The results of broad-spectrum assay showed that all five mAbs could react with H7 subtype AIV isolated in different years.The HI results showed that the HI titers of ascites against the H7-Re3 antigen strain were between 7 log2 and 12 log2.The results of the micro-neutralization test of MDCK cells showed that all five mAbs had neutralizing activity.Dot blot results showed that all five mAbs could recognize H7-Re2 antigenic strain and H7-Re3 variant strain.Western blot showed that a specific protein band appearing in the culture supernatant of five hybridoma cells was about 63 ku,indicating that these five mAbs recognized linear epitopes.In summary,five mAbs that broadly recognize the HA protein of H7 subtype AIV have been successfully prepared,which not only lays the foundation for the detection of influenza virus and the identification of neutralizing antibody epitopes,but also provides ideas for the design and immunological evaluation of new influenza vaccines.