猪流行性腹泻病毒N蛋白纳米抗体的筛选与鉴定  

Screening and Identification of Nanobodies Against Porcine epidemic diarrhea virus N Protein

作　　者：殷冬冬 丁祥 兰梦蝶 姬凯元 王洁茹 尹磊 沈学怀[1,2] 戴银 赵瑞宏[1,2] 候宏艳 胡晓苗[1,2] 潘孝成 YIN Dongdong;DING Xiang;LAN Mengdie;JI Kaiyuan;WANG Jieru;YIN Lei;SHEN Xuehuai;DAI Yin;ZHAO Ruihong;HOU Hongyan;HU Xiaomiao;PAN Xiaocheng(Livestock and Poultry Epidemic Diseases Research Center of Anhui Province,Institute of Animal Husbandry and Veterinary Science,Anhui Academy of Agricultural Sciences,Hefei 230001,China;Anhui Provincial Key Laboratory of Livestock and Poultry Product Safety Engineering,Hefei 230001,China;College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China;Ningguo City Animal Health Supervision Institute,Ningguo 242300,China)

机构地区：[1]安徽省农业科学院畜牧兽医研究所,安徽省畜禽疫病研究中心,安徽合肥230001 [2]畜禽产品安全工程安徽省重点实验室,安徽合肥230001 [3]安徽农业大学动物科技学院,安徽合肥230036 [4]宁国市动物卫生监督所,安徽宁国242300

出　　处：《华北农学报》2024年第6期224-230,共7页Acta Agriculturae Boreali-Sinica

基　　金：安徽省重点研究与开发计划项目(202204c06020009);安徽省自然科学基金项目(2308085MC102);安徽省农业科学院科研平台项目(2024YL016);安徽省现代农业产业技术体系(AHCYJXTX-05-13)。

摘　　要：由猪流行性腹泻病毒(PEDV)引起的高度接触性胃肠道传染病对我国养猪业造成巨大的经济损失。为建立PEDV抗体检测方法及N蛋白的功能研究奠定基础,通过噬菌体展示技术针对PEDV N蛋白进行筛选获得其特异性的纳米抗体(Nb)。利用前期制备的N蛋白免疫羊驼,采集其外周血并分离淋巴细胞,提取细胞总RNA,反转录为cDNA,通过PCR扩增重链抗体重链可变区(VHH),插入pCANTAB5E-ccdb载体,电转化至ER2738感受态细胞,构建VHH噬菌体展示文库;随后,以PEDV N蛋白作为靶向蛋白,对文库进行4轮淘选获得阳性噬菌体,将其克隆至pET-30a载体,表达纯化后通过间接ELISA、Western Blot鉴定Nb的结合力和特异性。结果显示,羊驼经5次免疫后,抗体效价达到1∶25600。构建的噬菌体展示文库库容为4.72×10^(8),丰度为4.3×10^(10 )cfu/mL,阳性率为93.75%。经过4轮筛选,获得16株氨基酸序列不同的Nb,随后验证了Nb45与PEDV N蛋白具有良好特异性和结合能力。研究筛选获得了PEDV N蛋白特异性Nb,为PEDV检测方法的建立和基础研究提供生物材料。The highly contagious gastrointestinal infectious disease caused by Porcine epidemic diarrhea virus(PEDV)has led to significant economic losses in China′s pig industry.It aimed to establish a basis for PEDV antibody detection methods and functional research of the N protein through the screening of specific nanobodies(Nbs)using phage display technology.Peripheral blood lymphocytes were isolated from a camel immunized with the N protein,and total RNA was extracted and reverse transcribed into cDNA.The variable domain of the heavy chain of heavy chain antibodies(VHH)was amplified by PCR,subcloned into the pCANTAB5E-ccdb vector,and electroporated into ER2738 competent cells to construct the VHH phage antibody display library.Subsequently,the library was subjected to four rounds of panning against the PEDV N protein,and positive phage clones were cloned into the pET-30a vector.The binding affinity and specificity of the Nbs were determined by indirect ELISA and Western Blot.The results showed that after the fifth immunization,the antibody titer reached 1∶25600.The constructed phage display library had a capacity of 4.72×10^(8) and an abundance of 4.3×10^(10) cfu/mL,with a 93.75%positive rate.After four rounds of screening,16 Nb clones with different amino acid sequences were obtained,and Nb45 was validated to possess excellent specificity and binding ability to the PEDV N protein.This study successfully screened and obtained specific N protein-targeting Nbs,providing biological materials for the establishment of PEDV detection methods and foundational research.

关 键 词：猪流行性腹泻病毒 N蛋白 噬菌体展示 纳米抗体 特性鉴定 

分 类 号：S858.285.3[农业科学—临床兽医学]