首页|鼠疫耶尔森菌低钙应答V抗原人源单克隆抗体筛选与鉴定

鼠疫耶尔森菌低钙应答V抗原人源单克隆抗体筛选与鉴定

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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× 108.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.
Screening and identification of human monoclonal antibodies against low-calcium response V antigen of Yersinia pestis
To characterize human antibodies against low-calcium response V(LcrV)antigen of Yersinia pestis,the mono-clonal antibodies were screened and assayed.Antibody gene was derived from peripheral blood mononuclear cells of the vaccin-ees immunized by plague subunit vaccine in phase Ⅱb clinical trial.Human ScFv antibody library was constructed by phage dis-play.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 estab-lished contained 7.54× 108 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 antibod-ies 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.

Yersinia pestislow-calcium response V antigenphage displayrecombinant antibodyprotective effect

张黎、郑滨洋、张琪、吴海莲、潘红星、朱凤才、吴海生、周剑芳

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中国疾病预防控制中心病毒病预防控制所,传染病溯源预警和智能决策全国重点实验室,北京 102206

江苏省疾病预防控制中心,南京 210009

青海省地方病预防控制所,西宁 810021

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

江苏省重点研发计划(社会发展)项目

BE2020720

2024

中国人兽共患病学报
中国微生物学会

中国人兽共患病学报

CSTPCD北大核心
影响因子:0.814
ISSN:1002-2694
年,卷(期):2024.40(1)
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