抗鼠疫菌保护性抗原V抗体的筛选与鉴定  

Screening and identification of monoclonal antibodies against protective V antigen of Yersinia pestis

作　　者：谢青 李志颖 张卫 郭家正 黄莺[1] 王茜 陈蕾 张艺潇 陆健昇[1] 杨志新[1] XIE Qing;LI Zhi-ying;ZHANG Wei;GUO Jia-zheng;HUANG Ying;WANG Xi;CHEN Lei;ZHANG Yi-xiao;LU Jian-sheng;YANG Zhi-xin(Institute of Biotechnology,Academy of Military Sciences,Beijing 100071,China)

机构地区：[1]军事科学院军事医学科学院生物工程研究所,北京100071

出　　处：《中国病原生物学杂志》2022年第3期266-271,共6页Journal of Pathogen Biology

摘　　要：目的构建纳米抗体噬菌体展示库,筛选抗鼠疫菌中和抗体,为鼠疫防治药物研制奠定基础。方法重组鼠疫菌保护性抗原V蛋白免疫双峰驼,分离外周血单核细胞,提取RNA,构建纳米抗体噬菌体展示库。以V蛋白为抗原对抗体库进行3轮固相筛选,挑选单克隆phage-ELISA鉴定,然后将富集的纳米抗体进行表达、纯化及评价。结果成功构建了库容量为1.9×10^(8)的抗鼠疫菌V蛋白纳米抗体噬菌体展示库。经过3轮筛选,获得8种抗体序列,表达和纯化后经ELISA检测,8株纳米抗体针对鼠疫菌V蛋白特异性良好且结合活性高。动物实验表明其中一株纳米抗体VHH-hFc6对鼠疫菌201株气溶胶攻击的保护率为33%。结论成功构建了抗鼠疫菌保护性抗原V蛋白的纳米抗体噬菌体展示库,筛选出8株抗V蛋白特异性纳米抗体,其中一株具有良好的中和活性。Objective To construct a Nanobody(Nb)phage display library to screen neutralizing antibodies against Yersinia pestis in order to facilitate the development of preventive and therapeutic drugs against plagues.Methods The bactrian camel was immunized with recombinant Yersinia pestis protective antigen V protein.Peripheral blood lymphocytes were isolated,total RNA was extracted,and a phage display library was constructed.Then,protein V was used as an antigen to screen the antibody library for three rounds,and phage-ELISA was used for monoclonal identification.The specificity and binding activity of the antibodies were identified by ELISA after the expression and purification of the enriched Nbs.And the neutralizing activity of the antibody in vivo was evaluated by the mouse protection experiment.Results The phage antibody library with effective library capacity of 1.9×10~8 and good diversity of nanobody against Y.pestis V protein was successfully constructed.Three rounds of screening were performed on the antibody library,and a 666-fold enrichment for specific phage antibodies was achieved.Through phage-ELISA identification and sequence analysis,two groups of phage Nanobody sequences with completely different sequences were obtained.They were successfully transformed into fusion proteins linked to human Fc,and were expressed and purified,named VHH-hFc-1~8 respectively.The eight nanobodies showed good specificities and high binding activity against Y.pestis V protein by ELISA..Further animal experiments showed that one of the nanoibodies named VHH-hFc6 could produce 33%protection against Y.pestis 201 aerosol attack.The EC_(50)value of this nanobody was determined by ELISA to be 2.452 ng/mL.Conclusion A nanobody library against V protein has been successfully constructed,and eight specific nanobodies against Y.pestis protective antigen V protein have been screened,one of which has good neutralizing activity.

关 键 词：鼠疫菌 V抗原 纳米抗体 中和抗体 

分 类 号：R378.6[医药卫生—病原生物学]