猪流行性腹泻病毒N蛋白纳米抗体的筛选与鉴定

doi:10.7668/hbnxb.20195229

华北农学报 ›› 2024, Vol. 39 ›› Issue (6): 224-230. doi: 10.7668/hbnxb.20195229

所属专题： 植物保护；畜牧；

• 畜牧·水产·兽医 • 上一篇下一篇

猪流行性腹泻病毒N蛋白纳米抗体的筛选与鉴定

殷冬冬¹^,², 丁祥³, 兰梦蝶⁴, 姬凯元³, 王洁茹¹^,², 尹磊¹^,², 沈学怀¹^,², 戴银¹^,², 赵瑞宏¹^,², 候宏艳¹^,², 胡晓苗¹^,², 潘孝成¹^,²

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

收稿日期:2024-06-21 出版日期:2024-12-28
通讯作者:
潘孝成(1971—),男,安徽霍邱人,博士,副研究员,主要从事畜禽病原微生物致病机制研究。
作者简介:
殷冬冬(1990—),男,安徽滁州人,博士,助理研究员,主要从事畜禽传染病防控研究。
基金资助:
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)

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

YIN Dongdong¹^,², DING Xiang³, LAN Mengdie⁴, JI Kaiyuan³, WANG Jieru¹^,², YIN Lei¹^,², SHEN Xuehuai¹^,², DAI Yin¹^,², ZHAO Ruihong¹^,², HOU Hongyan¹^,², HU Xiaomiao¹^,², PAN Xiaocheng¹^,²

¹ 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
² Anhui Provincial Key Laboratory of Livestock and Poultry Product Safety Engineering,Hefei 230001,China
³ College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China
⁴ Ningguo City Animal Health Supervision Institute,Ningguo 242300,China

Received:2024-06-21 Published:2024-12-28

摘要/Abstract

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。

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

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.

Key words: Porcine epidemic diarrhea virus, N protein, Phage display, Nanobody, Characterization

殷冬冬, 丁祥, 兰梦蝶, 姬凯元, 王洁茹, 尹磊, 沈学怀, 戴银, 赵瑞宏, 候宏艳, 胡晓苗, 潘孝成. 猪流行性腹泻病毒N蛋白纳米抗体的筛选与鉴定[J]. 华北农学报, 2024, 39(6): 224-230. doi: 10.7668/hbnxb.20195229.

YIN Dongdong, DING Xiang, LAN Mengdie, JI Kaiyuan, WANG Jieru, YIN Lei, SHEN Xuehuai, DAI Yin, ZHAO Ruihong, HOU Hongyan, HU Xiaomiao, PAN Xiaocheng. Screening and Identification of Nanobodies Against Porcine epidemic diarrhea virus N Protein[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(6): 224-230. doi: 10.7668/hbnxb.20195229.

http://www.hbnxb.net/CN/Y2024/V39/I6/224

图/表 7

表1 VHH片段引物序列

Tab.1 Primer sequences for VHH fragments

引物名称 Primer name	引物序列(5'—3') Primer sequences(5'—3')
Call001-F	GTCCTGGCTGCTCTTCTACAAGG
Call001-R	GGTACGTGCTGTTGAACTGTTCC
VHH-F	TTTCTATTACTA GGCCCAGCCGGCCAGKTGCAGCTCGTGGAGTCNGGNGG
VHH-R	AAACCGTT GGCCATAATGGCCTGGAGCTGGGGTCTTCGCTGTGGT

图1 羊驼血清中N蛋白抗体效价测定

Fig.1 Titer of anti-N antibody in alpaca

图2 VHH噬菌体展示文库的构建 A.第1轮PCR结果:M.DNA分子质量标准DL2000,1.阴性对照,2—4.IgG;B.第2轮PCR结果:M.DNA分子质量标准DL2000,1.阴性对照,2—4.VHH;C.PCR鉴定文库阳性率:M.DNA分子质量标准DL2000,1—48.48个不同的单克隆。

Fig.2 Construction of a VHH phage display library A.The first round of PCR:M.DL2000 DNA Marker,1.Negative control,2—4.IgG;B.The second round of PCR:M.DL2000 DNA Marker,1.Negative control,2—4.VHH;C.The library positivity rate was identified by PCR:M.DL2000 DNA Marker,1—48.Different monoclonals.

表2 4轮噬菌体淘洗的富集程度

Tab.2 Enrichment degree of four-round phage elutation

筛选轮数 Elutriation rounds	拯救噬菌体/ (cfu/mL) Input phage	洗脱噬菌体/ (cfu/mL) Output phage	回收率 Output/ Input
第1轮 First round	1.28×10¹³	4.50×10⁴	3.52×10^-9
第2轮 Second round	5.68×10¹³	5.30×10⁶	9.33×10^-8
第3轮 Third round	7.04×10¹³	2.90×10⁷	4.12×10^-7
第4轮 Fourth round	3.94×10¹²	1.46×10⁸	3.71×10^-5

图3 PEDV N蛋白特异性纳米抗体筛选 A.特异性噬菌体鉴定;B.PEDV N蛋白特异性纳米氨基酸序列比对。

Fig.3 Screening of specific nanobody of PEDV N protein A.Identification of specific phages;B.Alignment of Nb amino acid sequences.

图4 纳米抗体表达 A. Nb41和Nb44的表达:1,5. 16 ℃诱导Nb41、Nb44的上清,2,6. 16 ℃诱导Nb41的包涵体,3,7. 37 ℃诱导Nb41、Nb44的上清,4,8. 37 ℃诱导Nb41、Nb44的包涵体;B. Nb45和Nb46的表达: 1,5. 16 ℃诱导Nb45、Nb46的上清,2,6. 16 ℃诱导Nb45、Nb46的包涵体,3,7. 37 ℃诱导Nb45、Nb46的上清,4,8. 37 ℃诱导Nb45、Nb46的包涵体;C. Nb47和Nb96的表达: 1,5. 16 ℃诱导Nb47、Nb96的上清,2,6. 16 ℃诱导Nb47、Nb96的包涵体,3,7. 37 ℃诱导Nb47、Nb96的上清,4,8. 37 ℃诱导Nb47、Nb96的包涵体;D. Nb45的纯化: 1. 裂解液,2. 穿流液,3—4. 洗涤液,5—8. 洗脱液。

Fig.4 Expression of Nbs A. Expression of Nb41 and Nb44: 1,5. The supernatant of Nb41, Nb44 was induced at 16 ℃, 2,6. Inclusion bodies of Nb41, Nb44 were induced at 16 ℃, 3,7. Supernatant of Nb41, Nb44 was induced at 37 ℃, 4,8. Inclusion bodies of Nb41, Nb44 were induced at 37 ℃; B. Expression of Nb45 and Nb46: 1,5. The supernatant of Nb45, Nb46 was induced at 16 ℃, 2,6. Inclusion bodies of Nb45, Nb46 were induced at 16 ℃, 3,7. Supernatant of Nb45, Nb46 was induced at 37 ℃, 4,8. Inclusion bodies of Nb45, Nb46 were induced at 37 ℃; C. Expression of Nb47 and Nb96: 1,5. The supernatant of Nb47,Nb96 was induced at 16 ℃, 2,6. Inclusion bodies of Nb47,Nb96 were induced at 16 ℃, 3,7. Supernatant of Nb47,Nb96 was induced at 37 ℃, 4,8. Inclusion bodies of Nb47,Nb96 were induced at 37 ℃; D. Purification of Nb45: 1. Lysate, 2. Draining liquid, 3—4. Washing liquid, 5—8. Eluent.

图5 Nb45的特异性与结合力测定 A.Nb45的特异性检测;B.Nb45的结合力检测。

Fig.5 Binding capacity and specificity of Nb45 A.Specificity detection of Nb45;B.Binding capacity of Nb45.

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