全人源抗狂犬病病毒G蛋白单链抗体制备及中和活性鉴定  

Screening and preparation of a human single chain antibody(scFv)against Rabies Virus G protein from a phage display library and identification of the neutralizing activity

作　　者：张倩倩[1,2] 赵茜[3] 张晓[1] 丁贵鹏[1,2] 金秋[1] 高畅[2] 熊四平[2] 陈玉平 朱进[5] 冯振卿[1,2] 

机构地区：[1]南京医科大学卫生部抗体技术重点实验室,江苏南京210029 [2]南京医科大学病理学系,江苏南京210029 [3]杭州市红十字会医院结核科,浙江杭州310003 [4]江阴力博医药生物技术有限公司,江苏江阴214400 [5]南京军区军事医学研究所,江苏南京210002

出　　处：《南京医科大学学报（自然科学版）》2013年第7期927-931,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金资助(81273325);江苏省科技支撑计划资助(BE2011842)

摘　　要：目的:构建全人源抗狂犬病病毒scFv噬菌体抗体库,筛选针对狂犬病病毒G蛋白不同表位的单链抗体(scFv)并鉴定其中和活性。方法:分离130例经狂犬病疫苗免疫接种志愿者的外周血淋巴细胞,提取总RNA,逆转录制备cDNA,构建全人源抗狂犬病病毒scFv噬菌体抗体库。以纯化的狂犬病病毒G蛋白包板筛选,对阳性克隆进行可溶性表达,并鉴定中和活性。结果:构建全人源抗狂犬病病毒scFv噬菌体抗体库,库容为5.0×107,经核酸序列分析,证实插入片段为scFv。经过5轮筛选,从富集的次级抗体库中随机挑出140个克隆,通过phage-ELISA鉴定得到4株核酸序列不同的scFv抗体。经His-Trap纯化后进行中和活性鉴定,获得1株有中和活性的scFv,其中和效价为0.13 IU/mg。结论:构建全人源抗狂犬病病毒scFv噬菌体抗体库,获得1株具有中和活性的抗狂犬病病毒G蛋白scFv抗体,为进一步研发狂犬病治疗性抗体药物奠定了基础。Objective：To construct a human scFv phage antibody library against rabies virus G protein,selected the single chain antibody against rabies virus G protein for different epitopes,and identificated its neutralizing activity.Methods：The human scFv phage antibody library against rabies virus G protein was constructed by using peripheral blood lymphocytes from 130 Rabies vaccine immune volunteers.Screening by the purified rabies virus G protein,the positive clones were identified and expressed,identified neutralizing activity by AMMS.Results：Through the prokaryotic expression system and purification of rabies virus G protein,the human anti rabies virus G protein scFv phage antibody library was constructed with about 5.0×10 7 size.The nucleic acid sequence analysis confirmed the insertion of a fragment is scFv.After 5 rounds of screening,we randomly picked 140 clones by phage-ELISA, and obtained 4 nucleic acid sequences in different scFv antibodies,purified and identified activity.We finally obtained 1 scFv antibody with neutralizing activity.Conclusion：We have successfully constructed a whole human anti-rabies virus G protein scFv antibody library,and obtained a whole human anti-rabies virus G protein scFv antibody with neutralizing activity from the library.It is established foundation of the new type of rabies virus vaccine for human immunization.

关 键 词：狂犬病病毒G蛋白 噬菌体抗体库 单链抗体(scFv) 中和活性 

分 类 号：R392.12[医药卫生—免疫学]