摘要
目的制备和筛选抑制伯基特淋巴瘤(Burkitt lymphoma,BL)的抗EBV潜伏膜蛋白1(latent membrane protein-1,LMP1)单克隆抗体。方法人工合成EBV潜伏膜蛋白1的跨膜结构域5(transmembrane domain 5,TMD5),并以此为抗原,采用杂交瘤法制备抗TMD5单抗。ELISA法测定小鼠腹水中的抗体效价。7-氨基放线菌素D(7-Aminoactinomycin D,7-AAD)/Annexin V-PE双标记流式细胞术和JC-1染色联合流式细胞术测定线粒体膜电势评估单抗对BL细胞系Daudi细胞的促凋亡活性。蛋白质印迹法测定Daudi细胞内促存活信号通路p38-MAPK/IKK/NF-κB相关蛋白的表达水平。结果经过2轮筛选,获得R2-2-5E-6C和R2-2-8D-3A两种单抗,这2种单抗均能与TMD5发生抗体-抗原特异性结合,而与GAPDH无免疫反应。与NC组相比,R2-2-5E-6C组和R2-2-8D-3A组处理后的Daudi细胞中Annexin V+7-AAD+细胞所占百分比增加;JC-1荧光强度<104的细胞所占百分比增加;胞内p38-MAPK、IKK和NF-κB的表达水平降低(P<0.05)。结论筛选获得的抗TMD5单抗R2-2-5E-6C和R2-2-8D-3A可通过抑制LMP1介导的p38-MAPK/IKK/NF-κB信号通路的活性促进BL凋亡。
Objective To prepare and screen out the anti-EBV(Epstein-Barr virus)latent membrane protein-1 monoclonal antibodies against Burkitt’s lymphoma(BL).Methods EBV latent membrane protein-1(LMP1)transmembrane domain 5(TMD5)was synthesized,and was used as the antigen to prepare the anti-TMD5 monoclonal antibodies by hybridoma method.ELISA assay was used to determine the titer of antibodies in mouse ascites.7-AAD/Annexin V-PE in combination of flow cytometry(FCM)and JC-1 staining in combination of FCM were used to evaluate the apoptotic activity of BL cell line Daudi cells after treatment of monoclonal antibodies.Western blotting assay was used to determine the expression levels of p38-MAPK,IKK,NF-κB.Results After 2 rounds of screening,monoclonal antibodies(R2-2-5E-6C and R2-2-8D-3A)were obtained.Both of those could specifically bind to TMD5 peptide,but had no immune response to GAPDH.Compared with these in the NC group,the percentage of Annexin V+7-AAD+cells after treatment in the R2-2-5E-6C and R2-2-8D-3A groups were increased,the percentage of cells with JC-1 fluorescence intensity less than 104 were increased,and the expression levels of intracellular p38-MAPK,IKK and NF-κB were decreased in the Daudi cells(P<0.05).Conclusion The obtained anti-TMD5 monoclonal antibodies R2-2-5E-6C and R2-2-8D-3A could promote BL apoptosis through the inhibition of p38-MAPK/IKK/NF-κB pro-survival signaling pathway mediated by LMP1.
作者
涂小云
陈宇
熊玉红
邓爱花
孙春枝
刘阳
TU Xiaoyu;CHEN Yu;XIONG Yuhong;DENG Aihua;SUN Chunzhi;LIU Yang(Department of Infection and Immunization,Jiangxi Chest Hospital,Nanchang,330006,China)
出处
《医学分子生物学杂志》
CAS
2023年第6期467-472,共6页
Journal of Medical Molecular Biology
基金
江西省自然科学基金(No.20202BABL206085)。
