抑制EB病毒阳性弥漫性大B细胞淋巴瘤增殖的抗TMD5单克隆抗体的筛选和效果评价

Screening and efficacy evaluation of anti-TMD5 monoclonal antibodies against Epstein-Barr virus-positive diffuse large B-cell lymphoma

目的:筛选抗爱泼斯坦—巴尔病毒(EBV)潜伏期膜蛋白-1中跨膜结构域5(TMD5)单克隆抗体并评价其抑制EB病毒阳性弥漫性大B细胞淋巴瘤(DLBCL)的效果。方法:人工合成TMD5肽段,用此TMD5肽段免疫BALB/c小鼠;PEG法制备杂交瘤;ELISA法筛选阳性杂交瘤细胞;有限稀释法制备单克隆;使用阳性孔单克隆细胞免疫小鼠制备腹水;western blotting法和ELISA法分别对腹水中的单克隆抗体进行鉴定和效价测定;体外CCK-8法测定腹水对Sp2/0细胞存活能力的影响;Annexin VFITC/PI双标记流式细胞术测定腹水对Sp2/0细胞凋亡率的影响;在体内裸鼠荷瘤实验测定腹水对裸鼠荷瘤生长的影响。结果:成功备制13个杂交瘤,其中3个为阳性杂交瘤,分别为2B11、5D07和6F04,阳性率为23%。以5D07孔单克隆细胞制备的腹水以1∶105稀释后,阳性/阴性腹水单克隆抗体抗TMD5效价比为6.763,阳性/Sp2/0的效价比为5.952,效价最高。与PBS组相比,2B11组和5D07组DB细胞48 h和72 h增殖能力降低,凋亡率升高,裸鼠荷瘤较小(P<0.05),6F04组上述测定指标无明显差异(P>0.05)。与2B11组相比,5D07组DB细胞48 h和72 h增殖能力降低,凋亡率升高,裸鼠荷瘤较小(P<0.05)。结论:通过杂交瘤法筛选出的5D07单克隆抗体抗TMD5效价高,在体外和在体裸鼠荷瘤实验抗EBV+DLBCL淋巴瘤活性良好。

Objective:To screen the anti-transmembrane domain 5(TMD5)monoclonal antibodies of Epstein-Barr virus(EBV)latent membrane protein-1 and evaluate their inhibitory effect on monoclonal antibody against EBV positive diffuse large B-cell lymphoma(DLBCL).Methods:Synthesized TMD5 peptide fragment was used to immunize BALB/c mice.PEG method was used to prepare hybridomas.ELISA was used to screen the positive hybridoma cells.A limited dilution method was used to prepare monoclonal cells.Ascites was prepared by immunizing mice with positive monoclonal cells.Western blotting and ELISA were used to identify and titer determine the monoclonal antibodies in ascites,respectively.Cell counting kit-8(CCK-8)method was used to measure the effect of ascites on the viability of myeloma Sp2/0 cells in vitro.Annexin V-FITC/PI double-stained flow cytometry was used to determine the effect of ascites on the apoptosis rate of Sp2/0 cells.The effect of ascites on the tumor growth in nude mice was determined in vivo.Results:Thirteen hybridomas were successfully formed,of which 3 were positive,namely 2B11,5D07,and 6F04,with a positive rate of 23%.When the ascites prepared by 5D07 monoclonal cells were diluted at 1∶105,the titer ratio of positive/negative monoclonal antibody against TMD5 was 6.763,and the titer ratio of positive/Sp2/0 was 5.952,which had the highest titer.Compared with the PBS group,the proliferative ability of DB cells at 48 h and 72 h in 2B11 and 5D07 groups decreased,the apoptosis rate increased,and the nude mice tumors were smaller(P<0.05),while there was no significant difference was found in the parameters mentioned above in 6F04 group(P>0.05).Compared with the 2B11 group,the proliferative ability of DB cells at 48 h and 72 h in the 5D07 group decreased,and the apoptosis rate increased,while the nude mice tumors were smaller(P<0.05).Conclusion:The 5D07 monoclonal antibody screened by the hybridoma method has a high titer against TMD5 and has a good activity against EBV+DLBCL lymphoma in vitro and in vivo.