呼吸道合胞病毒 L 蛋白单克隆抗体的制备

目的：制备人呼吸道合胞病毒L蛋白单克隆抗体,并对其进行性质鉴定。方法：选取RSV-A2 L蛋白上具有亲水性可能的loop区段,将这些目的片段展示在HBc149上进行原核表达并纯化。重组蛋白免疫6~8周BALB/c雌性小鼠,根据免疫后血清的ELISA和WB分析结果选择L3935和L4099组小鼠进行脾脏免疫。融合后,用间接ELISA法筛选和多次克隆化,获得稳定分泌抗L蛋白单克隆抗体细胞株,并结合ELISA、免疫荧光和免疫印迹对所获得的单抗进行鉴定。结果：获得11株L蛋白特异性单克隆抗体,亚类鉴定结果IgG1、IgG2a和IgG3单抗各3株,IgG2b单抗2株。结论：成功制备了L蛋白特异性单克隆抗体,也为RSV感染的免疫和发病机制研究奠定基础。

Preparation of monoclonal antibodies against respiratory syncytial virus L protein

Objective:To prepare the monoclonal antibodies specifically against L protein of human respiratory syncytial virus,and to characterize these antibodies. Methods: The potential hydrophilic loop regions of RSV-A2 L protein were displayed on the vector HBc149 by using prokaryotic expression and purification. Recombinant protein was used to immunize female BALB/c mice for 6 to 8 weeks old,according to the results of ELISA and WB analysis,and the mice of group L3935 and L4099 were immunized by the spleen. And then,the hybridoma cells stably secreting McAb against L protein were selected by indirect ELISA detection.The monoclonal antibodies was identified by ELISA,immunofluorescence assay and immunoblotting analysis.Results:Eleven Lprotein specific monoclonal antibodies were obtained.Three mAbs were identified as IgG1 subtype,three as IgG2a,three as IgG3 and two as IgG2b. Conclusion:Lprotein specific monoclonal antibodies are successfully prepared,which will lay the foundation for the study of immunity and pathogenesis of RSV infection.