摘要
将增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,eGFP)免疫羊驼后构建纳米抗体免疫库,固相淘选eGFP纳米抗体;构建E.coli Rosetta表达载体,诱导表达纳米抗体,并鉴定其结合活性及特异性;辣根过氧化物酶(Horseradish peroxidase,HRP)标纳米抗体,确定其工作浓度及稳定性;将纳米抗体与纳米磁珠偶联应用于免疫沉淀B13-EGFP融合蛋白。四免后抗血清滴度超过1.28×10^(6),获得库容量为1.85×10^(8) cfu的抗eGFP纳米抗体噬菌体展示文库;原核表达10种独特基因序列的纳米抗体,均为可溶性表达;采用ForteBio Octet确定出纳米抗体4-28与eGFP的亲和常数(KD)最高,达到9.67×10^(-11)M;纳米抗体4-28与eGFP、绿色荧光蛋白(Green Fluorescent Protein,GFP)特异性结合;HRP标记纳米抗体4-28检测范围是1:10000~1:25000(浓度100~40 ng·mL^(-1)),比市场上现有的检测二抗(稀释范围1:5000~1:20000)灵敏度更高;纳米抗体4-28偶联羧基化纳米磁珠,免疫沉淀B13-eGFP融合蛋白,实现了简单快速分离目的蛋白B13-eGFP。
The peripheral blood of eGFP immunized alpaca was used to construct a nanobody immune library,and the eGFP nanobody was solid-phase panned.The E.coli Rosetta expression vector was constructed and expressed nanobody.The binding activity and specificity of nanobody were identified.The working concentration and stability of the enzyme-labeled nanobody was verified after HRP enzyme-labeled nanobody.The nanobody and nanomagnetic beads were coupled to obtain the B13-EGFP fusion protein by immunoprecipitation.After four immunizations,the antiserum titer exceeded 1.28×10^(6) and the eGFP nanobody library with a storage capacity of 1.85×10^(8) cfu was obtained.Nanobodies of ten unique gene sequences were expressed in prokaryotic cells,and they were all soluble intracellularly.Nanobodies 4-28 have the best affinity constant(KD)with eGFP by ForteBio Octet,reaching 9.67×10^(-11)M;Nanobody 4-28 specifically binds with eGFP and GFP.The detection range of the HRP enzyme-labeled nanobody 4-28 was 110000~125000(100~40 ng·mL^(-1)),which is higher than existing detection secondary antibodies on the market(dilution range 15000~120000).Nanobody 4-28 was coupled with carboxylated magnetic nanobeads to immunoprecipitate the B13-eGFP fusion protein,leading to a simple and rapid separation of the target protein B13-eGFP.
作者
刘晓芳
刘贝
周方月
黄云祥
钟引凤
张茹云
梁宇芳
王丹
付金衡
LIU Xiaofang;LIU Bei;ZHOU Fangyue;HUANG Yunxiang;ZHONG Yinfeng;ZHANG Ruyun;LIANG Yufang;WANG Dan;FU Jinheng(Institute of Life Sciences,Nanchang University,Nanchang 330031,China;Sino-Germany Joint Research Institute,Nanchang University,Nanchang 330047,China;Jiangxi Agricultural University,Nanchang 330045,China)
出处
《南昌大学学报（理科版）》
CAS
北大核心
2021年第1期34-40,共7页
Journal of Nanchang University(Natural Science)
基金
国家自然科学基金资助项目(31471648)。
