摘要
目的获得分泌特异性抗H1N1亚型(A/California/04/2009)猪流感病毒单克隆抗体(mAb)的杂交瘤细胞并探索其分泌抗体的功能。方法以表达血凝素蛋白H1(A/California/04/2009)的DNA疫苗为免疫原,免疫6～8周龄雌性BALB/c小鼠,取其脾细胞与NS1骨髓瘤细胞进行细胞融合,通过间接ELISA筛选分泌抗H1N1亚型(A/California/04/2009)抗体的杂交瘤细胞,进一步亚克隆筛选单一、稳定分泌其抗体的细胞株。结果获得能稳定分泌抗流感病毒血凝素H1亚型的单克隆抗体杂交瘤4株,分别命名为41H2、68E5、41E7和26D11。其中68E5、41E7和26D11为IgG1(K),41H2为IgG2a(K)。运用Lenti-pseudovirus方法测试显示,26D11可识别结构性位点,具有H1N1(A/California/04/2009)专一性较强的中和能力,是中和性抗体。Western blot检测显示,41H2和41E7为阳性,识别线性位点。特异性实验表明,26D11只与H1(A/California/04/2009)亚型流感病毒发生特异性反应,而不与其他16个HA亚型流感病毒发生交叉反应,显示出良好的特异性。结论用单克隆抗体杂交瘤技术成功制备了甲型H1N1流感病毒的单克隆抗体。
Objective Objective Preparing specialized anti-influenza A（H1N1） virus monoclonal antibodies,and charactering biological function of the antibodies.Methods Six to eight weeks female BALB/c mice were immunized with DNA vaccine which expresses hemagglutinin H1（A/California/04/2009）.Mosuse spleen B cells were fused with plasmocytoma cells NS1.Hybridoma cells were screened by indirect ELISA,then subcloning was done to obtain the cell line which secreted anti-influenza A（H1N1） virus monoclonal antibody in a specializing and steady way.Results Four monoclonal antibody cell lines were obtained which can steadily secret anti-influenza A（H1N1） virus hemagglutinin H1 subtype,named as 41H2,68E5,41E7 and 26D11.Among these antibodies,68E5,41E7 and 26D11 were charactered as IgG1;41H2 was charactered as IgG2a,all the antibodies＇ light chain was kappa.Lenti-pseudovirus test showed 26D11 could recogenize the constructive sites,had great ablity to neutralize H1N1（A/California/04/2009） and it was neutralizing antibody.41H2 and 41E7 were proved to be recogenizing the linear epitope by Western blot test.To showed 26D11 could only specialize reacting to influenza A virus H1 subtype,and there was no crosslink reaction to the other 16 HA subtype.Conclusion Specialized anti-influenza A（H1N1） virus monoclonal antibodies are generated.
出处
《苏州大学学报（医学版）》
CAS
北大核心
2011年第4期600-604,共5页
Suzhou University Journal of Medical Science
关键词
流感病毒
核酸疫苗
H1N1亚型
单克隆抗体
influenza virus
nucleic acid vaccine
H1N1 subtype
monoclonal antibody
