猪细小病毒NS1蛋白单链抗体库的构建及筛选

doi:10.16431/j.cnki.1671-7236.2025.07.028

中国畜牧兽医 ›› 2025, Vol. 52 ›› Issue (7): 3276-3285.doi: 10.16431/j.cnki.1671-7236.2025.07.028

猪细小病毒NS1蛋白单链抗体库的构建及筛选

张丽萌^1,2,3, 李闰婷¹, 宋月¹, 聂晓宁¹, 孔莉², 单靖微², 许盈盈², 王林青^1,2, 陈龙欣^1,2

1. 郑州师范学院分子生物学实验室, 郑州 450044;
2. 郑州师范学院生命科学学院, 郑州 450044;
3. 河南农业大学动物医学院, 郑州 450046

收稿日期:2024-11-12 出版日期:2025-07-05 发布日期:2025-07-01
通讯作者: 王林青, 陈龙欣 E-mail:wanglintsing@163.com;chen_longxin@163.com
作者简介:张丽萌，E-mail：zlmklmyt@163.com。
基金资助:
国家自然科学基金项目(32071447)；河南省科技攻关计划项目(232102311138)；河南省高校科技创新人才支持计划(22HASTIT041)；河南省高等学校重点科研项目(24B230008)；河南省大学生创新创业训练计划项目(国家级)(202312949004)

Construction and Screening of Single Chain Antibody Library of Porcine Parvovirus NS1 Protein

ZHANG Limeng^1,2,3, LI Runting¹, SONG Yue¹, NIE Xiaoning¹, KONG Li², SHAN Jingwei², XU Yingying², WANG Linqing^1,2, CHEN Longxin^1,2

1. Laboratory of Molecular Biology, Zhengzhou Normal University, Zhengzhou 450044, China;
2. School of Life Science, Zhengzhou Normal University, Zhengzhou 450044, China;
3. College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China

Received:2024-11-12 Online:2025-07-05 Published:2025-07-01

摘要/Abstract

摘要： 【目的】筛选特异性抗猪细小病毒(Porcine parvovirus，PPV)非结构蛋白NS1的单链抗体(scFv)，为后续研究PPV奠定基础。【方法】通过原核表达系统诱导表达并纯化PPV NS1蛋白，利用重组NS1蛋白进行动物免疫，检测血清效价水平后，采集小鼠脾脏提取总RNA并逆转录成cDNA，以其为模板扩增抗体重链可变区(variable domain of heavy chains，VH)和轻链可变区(variable domain of light chains，VL)序列；通过重叠延伸PCR扩增获得scFv基因，将其与酶切的pSEXRTL2噬菌体载体连接并电转化大肠杆菌XLⅠ-Blue感受态细胞，构建鼠源性抗PPV NS1的scFv抗体文库，并测定文库质量及库容量；利用噬菌体展示技术筛选对NS1蛋白具有特异性的scFv，通过ELISA和Western blotting检测抗原与阳性噬菌体克隆的亲和力。【结果】本试验成功表达纯化获得可溶性PPV NS1重组蛋白；构建的鼠源抗NS1 scFv文库的库容量为2.7×10⁷ CFU/mL，文库阳性率为87.5%；通过4轮“吸附-洗脱-富集”噬菌体筛选，最终获得2株与PPV NS1蛋白特异性结合的亲和力较高的scFv。【结论】本研究通过原核表达纯化重组蛋白NS1，成功构建鼠源抗PPV NS1蛋白的scFv噬菌体文库，利用噬菌体展示技术筛选获得了可与NS1重组蛋白特异性结合的scFv。试验结果为进一步研发抗PPV药物及诊断试剂提供依据。

关键词: 猪细小病毒(PPV); 单链抗体(scFv); NS1蛋白; 噬菌体文库

Abstract: 【Objective】 The purpose of this test was to screen the single chain antibody fragment (scFv) against the non-structural protein NS1 of Porcine parvovirus (PPV), which laid a foundation for the further study of PPV. 【Method】 The expression of PPV NS1 protein was induced by prokaryotic expression system and purified,and the recombinant NS1 protein was used for animal immunization，and serum titer level was detected.Total RNA was extracted from mouse spleen and reverse-transcribed into cDNA,which was used as template to amplify the sequences of variable domain of heavy chains (VH) and variable domain of light chains (VL).scFv gene was amplified by overlap PCR,and was connected to pSEXRTL2 phage vector and electrotransformed into E.coli XLⅠ-Blue competent cells.A murine scFv antibody library against PPV NS1 was constructed,and its quality and storage capacity were measured.Phage display technology was used to screen scFv specific to NS1 protein,and the affinity between the antigen and the positive phage clone was detected by ELISA and Western blotting. 【Result】 In this experiment,soluble PPV NS1 recombinant protein was successfully expressed and purified.The capacity of the murine anti-NS1 scFv library was 2.7×10⁷ CFU/mL,and the positive rate of the library was 87.5%.After four rounds of "adsorption-elution-enrichment" phage screening,two scFv strains with high affinity for PPV NS1 protein were obtained. 【Conclusion】 In this study,a murine scFv phage library against PPV NS1 protein was successfully constructed by prokaryotic expression and purification of recombinant protein NS1.scFv that could bind specifically to PPV NS1 recombinant protein was obtained by phage display technique.The results provided the basis for further research and development of anti-PPV drugs and diagnostic reagents.

Key words: Porcine parvovirus(PPV); single chain antibody fragment(scFv); NS1 protein; phage library

中图分类号:

S852.65^＋9.2

张丽萌, 李闰婷, 宋月, 聂晓宁, 孔莉, 单靖微, 许盈盈, 王林青, 陈龙欣. 猪细小病毒NS1蛋白单链抗体库的构建及筛选[J]. 中国畜牧兽医, 2025, 52(7): 3276-3285.

ZHANG Limeng, LI Runting, SONG Yue, NIE Xiaoning, KONG Li, SHAN Jingwei, XU Yingying, WANG Linqing, CHEN Longxin. Construction and Screening of Single Chain Antibody Library of Porcine Parvovirus NS1 Protein[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3276-3285.

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猪细小病毒NS1蛋白单链抗体库的构建及筛选

Construction and Screening of Single Chain Antibody Library of Porcine Parvovirus NS1 Protein

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