大容量天然鸡源噬菌体单链抗体库的构建  

Construction of a large naive chicken-derived phage antibody library

作　　者：范佳铭 龙丽瑾 刘文韬 翟康乐 宫雅楠[1] 胡源[1] 孟凡亮[1] 魏销玥 郭亚慧 赵雅坤 王婉婷 张建中[1] 闫笑梅[1] Fan Jiaming;Long Lijin;Liu Wentao;Zhai Kangle;Gong Yanan;Hu Yuan;Meng Fanliang;Wei Xiaoyue;Guo Yahui;Zhao Yakun;Wang Wanting;Zhang Jianzhong;Yan Xiaomei(National Key Laboratory of Intelligent Tracing and Forecasting of Infectious Diseases,Chinese Center for Disease Control and Prevention,Beijing 102206,China;China Medical University,Shenyang 110122,Liaoning,China;Department of Gastroenterology,Peking University Third Hospital,Beijing 100191,China;Bayannur Prefectural Center for Disease Control and Prevention,Bayannur 015000,Inner Mongolia,China)

机构地区：[1]中国疾病预防控制中心传染病预防控制所,传染病溯源预警与智能决策全国重点实验室,北京102206 [2]中国医科大学,辽宁沈阳110122 [3]北京大学第三医院消化科,北京100191 [4]巴彦淖尔市疾病预防控制中心,内蒙古巴彦淖尔015000

出　　处：《疾病监测》2024年第4期466-471,共6页Disease Surveillance

基　　金：首都卫生发展科研专项(No.2024-2G-4363);国家自然科学基金面上项目(No.81873959);财政专项基金(No.102393230020020000002;No.102393220020020000031)。

摘　　要：目的本研究拟构建大容量天然鸡源噬菌体单链抗体库,用于传染病诊断用抗体筛选。方法选取未经免疫的5周龄白来航鸡包括普通级别和无特定病原体级别、罗曼鸡、洛岛红鸡、洛岛白鸡和贵妃鸡,每个品种各5只,共30只。从法氏囊中提取总RNA,通过反转录合成cDNA。利用聚合酶链式反应技术扩增全套编码抗体可变区的基因序列。通过重叠延伸反应,将重链可变区和轻链可变区基因连接成为单链抗体。将经过回收并酶切纯化的单链抗体克隆到噬菌体载体上,电转化大肠埃希菌TG1感受态细胞。结果成功构建库容量达1010的鸡源天然单链抗体库细胞库,噬菌体展示库滴度达1012菌落形成单位/mL以上。随机挑选165个单克隆进行菌落PCR鉴定,转化阳性插入率为96.97%(160/165);随机选取72株阳性克隆进行测序,抗体互补决定区3碱基序列及长度存在显著差异,表明构建的抗体库多样性良好。结论本研究成功构建了大容量鸡源天然单链抗体库,为后续从中筛选出具有应用价值的特异性单链抗体奠定了基础。Objective To construct a large naive chicken-derived phage antibody library for antibody screening in infectious disease diagnosis.Methods Five five-week old White Leghorn chickens,including ordinary ones and specific pathogen free ones,Roman chicken,Los Angeles Red chickens,Los Angeles White Chickens and Imperial chickens were selected respectively.Total RNA was extracted from bursa of Fabricius of 30 non-immunized chickens,then reversely transcribed to cDNA,and amplified by polymerase chain reaction to obtain the gene encoding the variable region of antibody.Splicing by overlap extension was used to assemble the gene coding the single-chain variable fragment.By ligation with the phage vector,the recombinant phage was electroporated into competent cells of Escherichia coli TG1 to construct a naive chicken-derived phage antibody library.Results The naive antibody library with capacity of 1010 was successfully constructed.The titer of phage display library exceeded 1012 colony forming units/mL.A total of 165 clones were randomly selected,resulting in a positive transformation insertion rate of 96.97%(160/165).Seventy-two positive clones were randomly selected for sequencing,and significant variations in the base sequence and length of the complementarity determining region 3 were found,indicating the good diversity of antibody library.Conclusion A naive single chicken-derived phage antibody library was successfully constructed in this study,which is fundamental for the valuable single chain antibody screening for specific antigens.

关 键 词：鸡源 天然抗体库 单链抗体 噬菌体展示技术 

分 类 号：R211[医药卫生—中医学] R392.11