A型流感病毒PA蛋白纳米抗体的筛选与鉴定

doi:10.16431/j.cnki.1671-7236.2025.12.028

摘要/Abstract

摘要： 【目的】构建A型流感病毒(Influenza A virus，IAV)聚合酶酸性蛋白(polymerase acidic protein,PA)的特异性纳米抗体(nanobody,Nb)噬菌体展示文库，获得广谱特异性识别PA蛋白的纳米抗体，为IAV基础研究和应用研究提供生物材料。【方法】构建真核表达IAV PA的重组质粒，使用Flag标签抗原特异抗体亲和层析法获得纯化蛋白，并以此作为免疫原与弗氏佐剂混合，对羊驼进行免疫。第5次免疫14 d后，采用间接ELISA检测羊驼血清中针对PA蛋白的抗体效价。从羊驼外周血中分离出淋巴细胞(peripheral blood lymphocyte,PBL)，提取总RNA后反转录成cDNA，利用巢式PCR技术扩增重链抗体的重链可变区(variable domain of heavy chain of heavy-chain antibody,VHH)基因，构建pComb-VHH噬菌体重组质粒并电转化大肠杆菌SS320感受态细胞，构建纳米抗体噬菌体展示文库，同时测定该文库库容量及多样性。使用PA蛋白包被酶标板，淘选获得与PA蛋白反应的纳米抗体阳性克隆。对淘选获得的阳性克隆进行测序及真核表达，通过Western blotting、间接免疫荧光试验鉴定纳米抗体特异性。【结果】经测序验证，获得序列正确的pcDNA3.1-Flag-PA真核表达重组质粒，表达并纯化获得PA蛋白作为免疫原对羊驼进行免疫。经5次免疫后，羊驼血清中针对PA蛋白的抗体效价达到1∶64 000；成功扩增出VHH基因并构建重组率为100%、序列多样性为90%、库容量为6.5×10⁷ CFU/mL的纳米抗体噬菌体展示文库。经过3轮富集淘选，获得6株功能区序列不同的PA蛋白纳米抗体，分别为Nb13、Nb14、Nb16、Nb20、Nb42和Nb72，其中纳米抗体Nb16可广谱特异性识别不同亚型IAV的PA蛋白。【结论】本研究成功构建 IAV PA蛋白纳米抗体噬菌体展示文库，筛选获得1株广谱特异性识别 IAV PA蛋白的纳米抗体。

关键词: A型流感病毒(IAV); 聚合酶酸性蛋白; 纳米抗体噬菌体展示文库; 纳米抗体

Abstract: 【Objective】 The goal of this study was to develop a broad-spectrum nanobody (Nb) specifically targeting the polymerase acidic protein (PA) of Influenza A virus (IAV),offering novel biomaterials for both basic and applied research of IAV. 【Method】 The eukaryotic expression plasmid of IAV PA was constructed,and purified proteins were obtained using Flag tag antigen-specific antibody affinity chromatography.It was mixed with Freund’s adjuvant to immunize alpacas.The titer of antibody against IAV PA in alpaca serum collected at 14 days post the fifth immunization was determined by indirect ELISA.Peripheral blood lymphocytes (PBL) were isolated from the peripheral blood of alpacas,and total RNA was extracted and reverse transcribed into cDNA.Following nested PCR amplification,the gene coding for the variable domain of heavy chain of heavy-chain antibody (VHH) was inserted into a pComb phage vector to construct a phage display library.The capacity of the library was calculated and its diversity was analyzed.Positive Nb clones binding to PA protein were selected through enrichment and panning.The specificity of Nbs was evaluated by Western blotting and immunofluorescence assay (IFA). 【Result】 After sequencing verification,the pcDNA3.1-Flag-PA eukaryotic expression recombinant plasmid with correct sequence was obtained.The PA protein was expressed and purified to be used as an immunogen for immunizing alpacas.After five immunizations,the antibody titer against PA protein in alpaca serum reached 1∶64 000. VHH gene was successfully amplified and a nanobody phage display library with a recombination rate of 100%,sequence diversity of 90%,and a library capacity of 6.5×10⁷ CFU/mL was constructed.After three rounds of enrichment and panning,six PA protein nanobodies with different functional region sequences were obtained,namely Nb13,Nb14,Nb16,Nb20,Nb42 and Nb72,respectively.Among them,the nanobody Nb16 could specifically recognize PA proteins of different subtypes of IAV in a broad-spectrum. 【Conclusion】 In this study,a phage display library of IAV PA protein nanobodies was successfully constructed,and a broad-spectrum nanobody specifically recognizing IAV PA protein was screened and obtained.

Key words: Key words:Influenza A virus (IAV); polymerase acidic protein; nanobody phage display library; nanobody

中图分类号:

S852.65

徐洁, 申汶涛, 朱启运, 徐帅. A型流感病毒PA蛋白纳米抗体的筛选与鉴定[J]. 中国畜牧兽医, 2025, 52(12): 5839-5848.

XU Jie, SHEN Wentao, ZHU Qiyun, XU Shuai. Screening and Identification of Nanobodies Against Polymerase Acidic Protein of Influenza A Virus[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(12): 5839-5848.

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