抗鸡传染性法氏囊病病毒单链抗体库的构建及其中和抗体的筛选  

Construction and screening of an anti-IBDV scFv library

作　　者：周兵[1] 李天鹤[2] 李宁[3] 徐黎明[4] 郭茉[1] 马蕾[1] 张瑛杰[1] 叶贤龙[1] 赵景壮[4] 衣春霖[1] 韩晓辉[1] 丁良君[1] 李德山[1] 

机构地区：[1]东北农业大学生命科学学院/生物制药教研室,黑龙江哈尔滨150030 [2]中国科学院大学生命科学学院,北京100049 [3]国家知识产权局专利局专利审查协作北京中心,北京100083 [4]中国水产科学研究院黑龙江水产研究所,黑龙江哈尔滨150070

出　　处：《中国预防兽医学报》2014年第3期227-231,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金青年科学基金项目(31001084);黑龙江省博士后科研启动基金项目(LBH-Q09162)

摘　　要：为构建抗鸡传染性法氏囊病病毒（IBDV）的细菌展示单链抗体（scFv）库并筛选出具有高亲和力和中和活性的scFv。本研究从IBDV免疫鸡的法氏囊提取总RNA，采用RT-PCR的方法分别扩增鸡抗体的VH和VL编码序列，插入含有（Gly4Ser）3 linker的pTlinker载体中，再将VH-linker-VL片段亚克隆到细菌展示载体pBSD中。以FITC标记的VP2 蛋白为诱饵，通过流式细胞术筛选出5株阳性scFv，其氨基酸同源性为82.16 %～87.27 %。将5株scFv基因分别插入原核表达载体pET-27b（＋）中，构建重组表达质粒pET-IBDV-scFv。将其转化大肠杆菌Rosetta进行诱导和表达，该5株scFv相对分子质量均约为28 ku。此外，利用纯化的scFv包被于ELISA检测板中作为扑捉抗体，检测结果显示scFv对VP2蛋白和不同IBDV株均具有特异性结合能力。而且通过体外中和试验结果表明，仅有一株scFv具有病毒中和活性，可以有效阻断IBDV（B87 株）对鸡胚成纤维细胞（DF1）的感染。该研究结果为进一步研制IBDV治疗性抗体奠定了基础。In this study, the bacteria-displayed recombinant scFv libraries were constructed from immunized chickens, five VP2-binding scFv clones with unique sequences were obtained after screening the scFv library by FCM with FITC-labeled VP2. The amino acid homology among scFvs were 72.46%. Five VP2-binding scFv clones were expressed in E.coli and purified through denaturation and renaturation of inclusion body. ELISA results showed that all scFvs exhibited binding ability and specifity to VP2 and various IBDV strains. In addition, one scFv showed significant neutralizing activity to IBDV （B-87 strain） as the scFv inhibited cytopathic effect of chicken embryo fibroblast （DF1） caused by IBDV. The study provides a lead candidate for further development of therapeutic antibodies for IBDV infection.

关 键 词：IBDV 细菌展示 单链抗体 中和活性 

分 类 号：S852.4[农业科学—基础兽医学]