【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∶25 600。构建的噬菌体展示文库库容为4.72×10⁸,丰度为4.3×10¹⁰ 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。更多还原

【Abstract】 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∶25 600.The constructed phage display library had a capacity of 4.72×10⁸ and an abundance of 4.3×10¹⁰ 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.更多还原