摘要
为获得抗O型口蹄疫病毒(FMDV)高亲和力猪源单链抗体(scFv),建立抗原抗体(Ag-Ab)共表达细菌展示文库,抗原为FMDV结构蛋白VP1、抗体为高免猪抗体文库。采用细菌展示与流式细胞术结合方法,筛选出14株抗FMDV的scFv。将筛选获得scFv构建至pET28a载体中,经E.coli Rosetta表达,目的蛋白长度约为30 ku,与scFv分子质量相符。Western blot结果表明,14株scFv均可与VP1特异性结合。ELISA结果表明,14株scFv均可与灭活O型FMDV特异性结合。应用共表达细菌展示技术,可避免抗原制备,简化操作流程,适用范围广泛;应用流式细胞仪筛选抗体库,可实现准确、快速、高通量筛选。抗FMDV猪源scFv研究可填补同源基因工程抗体空白,为进一步筛选中和抗体奠定基础。
In order to obtain a high affinity porcine single chain antibody(scFv)against the foot-and-mouth disease virus(FMDV),a bacterial display library co-expressing antigen-antibody(Ag-Ab)was constructed,and the antigen was FMDV structural protein VP1,and antibodies were derived from immunized pig.Total 14 anti-FMDV scFv were screened by bacterial display and flow cytometry.The scFv obtained by screening was constructed into pET28a vector and expressed by E.coli Rosetta,and the target protein size was about 30 ku,which was agreed with the molecular weight of scFv.Western blot analysis showed that 14 scFv could specifically bind to VP1.ELISA results showed that 14 scFv could specifically bind to inactivated O-type FMDV.The application of co-expression bacterial display technology could avoid the preparation of antigen,simplify the operation process,and had a wide range of applications;the application of flow cytometry to screen antibody libraries could achieve accurate and rapid high-throughput screening.The study of anti-FMDV porcine single-chain antibody filled the gap of homologous genetically engineered antibodies and laid the foundation for further screening of neutralizing antibodies.
作者
李德山
胡爽
赵文漾
李青青
郭笑辰
王丹
任桂萍
尹杰超
LI Deshan;HU Shuang;ZHAO Wenyang;LI Qingqing;GUO Xiaochen;WANG Dan;REN Guiping;YIN Jiechao(School of Life Sciences,Northeast Agricultural University,Harbin 150030,China)
出处
《东北农业大学学报》
CAS
CSCD
北大核心
2020年第1期42-49,共8页
Journal of Northeast Agricultural University
基金
国家重点研发计划项目（2016YFD0501003）
关键词
口蹄疫
单链抗体
细菌展示
流式细胞术
foot-and-mouth disease
single-chain antibody
bacterial display
flow cytometry
