H1N1型流感病毒小鼠杂交瘤单克隆抗体的制备和鉴定

Preparation and identification of monoclonal antibody against H1N1 influenza virus

目的纯化H1N1流感病毒作为抗原免疫小鼠制备单克隆抗体(mAb),并以mAb为工具分析病毒对宿主细胞的感染情况。方法鸡胚培养A/PR/8(H1N1),收获尿囊液,蔗糖密度梯度离心纯化病毒,透射电镜鉴定病毒颗粒,甲醛灭活病毒后免疫小鼠,评价抗原免疫效果,取鼠脾细胞与Sp2/0细胞融合制备mAb。用ELISA、免疫荧光细胞化学染色、Western blot法、血凝抑制试验和微量中和试验对mAb特性进行鉴定。依据流式细胞术用血凝素mAb分析流感病毒感染MDCK细胞后血凝素蛋白在宿主细胞膜上展示特征和病毒感染对细胞凋亡的影响。利用血凝素mAb建立基于细胞的ELISA(Cell-based ELISA)并对病毒增殖和感染动态进行分析。结果纯化出的A/PR/8病毒在电镜下呈圆形、椭圆形和棒状,免疫小鼠血清中流感病毒IgG滴度动态增长,免疫6周后IgG滴度达106。免疫小鼠脾细胞与Sp2/0细胞融合制备出6株流感病毒mAb,其中PR8-10mAb血凝抑制活性和中和活性均最高,分别为1∶2048和1∶640。免疫荧光细胞化学染色和Western blot结果表明该mAb可与血凝素结合。流式细胞术显示PR8-10也可识别细胞膜上的血凝素,同时发现病毒感染细胞后会引起细胞凋亡。依据PR8-10可识别细胞膜上的血凝素原理建立的Cell-based ELISA可分析病毒增殖情况。结论纯化出完整病毒颗粒免疫小鼠可刺激产生抗病毒IgG,并制备出高亲和活性和中和活性的H1N1病毒mAb,该mAb可分析病毒感染特征以及病毒对细胞的影响。

Objective To prepare the monoclonal antibody（m Ab） against influenza A virus（H1N1） using purified viral particles as antigen and investigate the characterization of host cel s infected with influenza virus utilizing the m Ab.Methods A/PR/8（H1N1） virus was cultured in embryonated chicken eggs and further purified by differential and density gradient centrifugation.The structure of viral particles was identified by transmission electron microcopy（TEM）.Immunogenicity of purified virus was evaluated by Balb/c mice immunized with formalin-inactivated virus.Hybridomas secreting m Abs were established through a fusion of Sp2/0 myeloma cel s and splenocytes from the mice immunized with the virus.The characteristics of m Ab were identified by ELISA,immunofluorescence assay（IFA）,Western blotting,hemagglutinin inhibition assay（HI） and microneutralization assay.The outside hemagglutinin（HA） on the plasma membrane of the host MDCK cells in which the viruses were propagated and the apoptosis of MDCK cells infected with the viruses were measured using flow cytometry.Cell-based ELISA was established using m Ab specific to HA.Subsequently,the growth of virus was analyzed by cell-based ELISA.Results Transmission electron microscopy revealed that the physical structures of the purified virus were spherical,elliptical and extended threadlike.Serum Ig G titer to influenza virus showed a progressive increase,and the Ig G titer reached106 after the immunization for 6 weeks.Six hybridoma clones secreting m Ab specific to A/PR/8 were developed by hybridoma technology.The HI and neutralization activities of PR8-10 m Ab were significantly higher than those of the other m Abs.HI and neutralization titers of PR8-10 m Ab were 1∶2048 and 1∶640,respectively.IFA and Western blotting confirmed that PR8-10 m Ab could recognize HA.Flow cytometry showed that PR8-10 m Ab also recognized HA on the membrane of MDCK in which the viruses were replicated and virus infection induced the apoptosis of MDCK cells.Based on the previous test results that PR8-10 m Ab was able to recognize HA on the membrane of the host cells in which the viruses were replicated,cell-based ELISA we established was good at analyzing the growth of virus in MDCK cells.Conclusion We obtained whole viral particles that were demonstrated to be able to stimulate the production of a high Ig G titer in a mouse model with formalin-inactivated viral particles,and successfully prepared the m Ab against H1N1 of high binding affinity and neutralization potency.HA-specific m Ab can be used to analyze the characteristics of virus infection process and the effect of virus infection on the host cel s as well.