抗鼠疫抗原F1的嵌合抗体制备及特性分析  

Preparation and characterization of chimeric antibody against plague antigen F1

作　　者：匡衡 蔡欣 杨展 周东明 周婷婷 汪茂荣 朱进 KUANG Heng;CAI Xin;YANG Zhan;ZHOU Dongming;ZHOU Tingting;WANG Maorong;ZHU Jin(Department of Hepatology,General Hospital of Eastern Theater Command of PLA,Bayi Clinical College Affiliated to Anhui Medical University,Nanjing 210001;Center of Eastern Theater for Disease Control and Prevention,Nanjing 210002,China)

机构地区：[1]东部战区总医院秦淮医疗区肝病科教研室,安徽医科大学八一临床学院,江苏南京210001 [2]东部战区疾病预防控制中心,江苏南京210002

出　　处：《南京医科大学学报（自然科学版）》2021年第12期1753-1758,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家生物安全专项(2018YFC1200603);江苏省社会发展项目(BE2018617)。

摘　　要：目的:建立抗鼠疫F1嵌合抗体的稳定表达细胞系,表达并纯化嵌合抗鼠疫F1抗体并鉴定其活性。方法:提取鼠源杂交瘤细胞株4C6总核糖核酸(ribonucleic acid,RNA),逆转录为互补脱氧核糖核酸(complementary DNA,c DNA),通过聚合酶链式反应(polymerase chain reaction,PCR)获得抗F1抗体轻链、重链可变区基因,全基因合成嵌合抗体的重链和轻链基因,并克隆于真核表达载体p MH3质粒中,共转染入中国仓鼠卵巢上皮细胞-S(Chinese hamster ovary cell-S,CHO-S)中,经遗传霉素(geneticin,G418)筛选得到稳定表达F1嵌合抗体的细胞株,悬浮培养后收集上清并亲和纯化。采用酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)方法检测其与F1抗原的特异性结合能力;通过竞争性ELISA比较嵌合抗体和鼠源亲本抗体与抗原的结合能力,同时测定抗体亲和力和质谱分析抗体特性。结果:成功获得稳定分泌抗鼠疫抗原F1嵌合抗体的稳定表达细胞系,该抗体能够特异性结合F1抗原,抗体的亲和力为2.303×10-9mol/L。结论:成功制备了抗体鼠疫F1的嵌合抗体,为后续动物体内实验及临床药物研发奠定了基础。Objective:This study aims to establish a stable cell line with high expression of anti-plague F1 chimeric antibody,express and purify the chimeric anti-plague F1 antibody and identify its activity.Methods:Total RNA of murine hybridoma cell line4 C6 was extracted and reverse transcribed into cDNA.The variable region genes of light chain and heavy chain of anti-F1 antibody were obtained by PCR and the gene synthesis chimeric antibody heavy chain and light chain gene,and cloning in eukaryotic expression vector pMH3 plasmid of transfection cells.CHO-S stable expression is obtained by G418 screening F1 chimeric antibody cell lines.Supernatant was collected and purified after suspension culture.The specific binding ability with F1 antigen was detected by ELISA.The binding ability of chimeric antibody and mouse parent antibody to antigen was compared by competitive ELISA,the antibody affinity was determined,and the antibody properties were analyzed by mass spectrometry.Results:Stable cell lines secreting chimeric antibody against plague antigen F1 were successfully obtained.The antibody could specifically bind to F1 antigen,and the affinity of the antibody was 2.303×10-9 mol/L.Conclusion:In this study,chimeric antibody against plague F1 was successfully prepared,which laid a foundation for subsequent animal experiments and clinical drug development.

关 键 词：鼠疫耶尔森菌 嵌合抗体 F1抗原 亲和力 质谱分析 

分 类 号：R392.11[医药卫生—免疫学]