摘要
目的筛选抗低钙应答V抗原(LcrV)的人源单克隆抗体,分析抗体基因特点,检测抗体与抗原结合的特异性、亲和力及功能。方法使用PCR方法从鼠疫疫苗临床试验志愿者外周血细胞cDNA扩增抗体轻、重链可变区基因片段,构建ScFv噬菌体抗体文库。用LcrV对文库进行富集筛选,将获得的抗体基因表达成人IgG1后,测定抗体与LcrV抗原结合的特异性、亲和力、免疫调节功能以及保护能力。结果成功构建了鼠疫耶尔森菌人源ScFv抗体文库,库容量为7.54×10^(8)。抗体文库经LcrV筛选后获得了3株抗LcrV抗体,命名为RV-B4、RV-D1和RV-E8。3株抗体重链为VH1-46和VH3-30,轻链分别为VL1-51、VK3-20和VK1-39。3株抗体经ELISA及Western blot验证均与LcrV特异性结合,其与V抗原结合的解离常数(KD)分别为2.1 nmol/L、1.24 nmol/L和42 nmol/L。RV-D1体外降低人THP-1分泌TNF-α,动物攻毒试验中未发现抗LcrV抗体的保护效果。结论从鼠疫疫苗免疫人群获得了靶向低钙应答V抗原的人源抗体,以结合抗体为主,不能有效阻断病菌感染。所获得的单抗为鼠疫免疫基础研究、鼠疫诊断应用提供了候选材料。
To characterize human antibodies against low-calcium response V(LcrV)antigen of Yersinia pestis,the monoclonal antibodies were screened and assayed.Antibody gene was derived from peripheral blood mononuclear cells of the vaccinees immunized by plague subunit vaccine in phase IIb clinical trial.Human ScFv antibody library was constructed by phage display.After panning library by using recombinant LcrV antigen,antibody variable genes were sequenced and converted into IgG1 format to evaluate its binding specificity and relevant parameters.An anti-plague human ScFv antibody library was established contained 7.54×10^(8)independent clones.After panning by LcrV antigen,3 human antibodies named as RV-B4,RV-D1 and RV-E8,respectively,were identified.Using indirect enzyme-linked immunosorbent assay(ELISA)and Western blot(WB),the specific bindings of the mAbs to LcrV antigen were confirmed.The dissociation constant(KD)of them to LcrV is 2.1 nmol/L,1.24 nmol/L and 42 nmol/L,respectively.Minor protective efficacy was found among 3 human antibodies in Y.pestis 141-infected mice.Three anti-LcrV monoclonal antibodies generated from immunized vaccinees were binding specific antibodies and could not block plague infection in mice.These antibodies are the potential candidate reagents for basic research of plague immunity and the application of plague diagnosis.
作者
张黎
郑滨洋
张琪
吴海莲
潘红星
朱凤才
吴海生
周剑芳
ZHANG Li;ZHENG Bin-yang;ZHANG Qi;WU Hai-lian;PAN Hong-xin;ZHU Feng-cai;WU Hai-sheng;ZHOU Jian-fang(National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases(NITFID),National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Jiangsu Provincial Center for Disease Control and Prevention,Nanjing 210009,China;Qinghai Institute for Endemic Disease Control and Prevention,Xining 810021,China)
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2024年第1期15-20,共6页
Chinese Journal of Zoonoses
基金
江苏省重点研发计划(社会发展)项目(No.BE2020720)。
关键词
鼠疫耶尔森菌
低钙应答V抗原
噬菌体展示
重组抗体
保护效果
Yersinia pestis
low-calcium response V antigen
phage display
recombinant antibody
protective effect
