猪流行性腹泻病毒S1蛋白的原核表达及其核酸适配体的筛选  

Prokaryotic Expression of S1 Protein in Porcine Epidemic Diarrhea Virus and Screening of Its Aptamers

作　　者：张冬萱 王智豪 乔岩 赵肖肖 范松杰 张超[1] ZHANG Dongxuan;WANG Zhihao;QIAO Yan;ZHAO Xiaoxiao;FAN Songjie;ZHANG Chao(Key Laboratory of Animal Biochemistry and Nutrition,Ministry of Agriculture and Rural Affairs,College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]河南农业大学动物医学院,农业农村部动物生化与营养重点开放实验室,郑州450046

出　　处：《畜牧兽医学报》2025年第2期839-850,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：河南省科技攻关(242102110019);国家自然科学基金(31972672)。

摘　　要：本文旨在可溶性原核表达猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)S1蛋白的C端结合域(S1-CTD),并利用指数富集的配体系统进化技术筛选靶向S1-CTD蛋白的DNA适配体,为该病毒治疗药物的开发以及检测方法的建立奠定基础。采用大肠杆菌原核表达系统,将PEDV-S1-CTD质粒与带有麦芽糖结合蛋白(MBP)、小分子泛素样修饰蛋白(SUMO)、反转录终止因子(NusA)、谷胱甘肽巯基转移酶(GST)四种促溶标签的原核表达载体pET21b连接,构建重组质粒pET21b-MBP-S1、pET21b-SUMO-S1、pET21b-NusA-S1、pET21b-GST-S1,将双酶切鉴定和测序正确的重组质粒转入大肠杆菌BL21进行诱导表达,选择可溶性好且表达量高的rMBP-S1重组蛋白进行后续试验,Western blot结果显示该重组蛋白正确表达,间接免疫荧光试验(IFA)结果表明rMBP-S1能与细胞表面受体结合。随后经磁珠法筛选得到111条靶向rMBP-S1重组蛋白的候选DNA适配体,其中Apt-S1-3、Apt-S1-11富集程度最高,分别是18%和16.2%,酶联适配体吸附试验ELASA(enzyme-linked aptamer sorbent assays,ELASA)测定适配体Apt-S1-3的解离常数为2.09 nmol,并且该适配体不与rMBP蛋白、rMBP-gD蛋白、rMBP-GP5蛋白以及BSA蛋白发生交叉反应,表明该适配体具有高亲和力与高特异性。此外,使用在线软件Mfold分析适配体Apt-S1-3形成的二级结构,并且通过分子对接模拟,发现该适配体能够与靶标蛋白形成稳定的复合物。最后的间接免疫荧光试验表明适配体Apt-S1-3能够识别PEDV,流式细胞术试验证实该适配体能够抑制PEDV感染宿主细胞。本研究筛选得到的DNA适配体能够与猪流行性腹泻病毒S1-CTD蛋白高亲和力、高特异性结合,而且能够抑制猪流行性腹泻病毒感染细胞,为PEDV的靶向治疗和检测方法开发奠定基础。This study aimed to express the C-terminal binding domain(S1-CTD)of the porcine epidemic diarrhea virus(PEDV)S1 protein in a soluble prokaryotic system and to screen DNA aptamers targeting S1-CTD using the systematic evolution of ligands by exponential enrichment(SELEX)technique,laying the foundation for the development of therapeutic drugs and detection methods for the PEDV.The PEDV-S1-CTD plasmid was cloned into prokaryotic expression vectors containing maltose-binding protein(MBP),small ubiquitin-like modifier(SUMO),transcription termination factor(NusA),and glutathione S-transferase(GST)tags.Recombinant plasmids pET21b-MBP-S1,pET21b-SUMO-S1,pET21b-NusA-S1,and pET21b-GST-S1 were constructed and transformed into E.coli BL21 for expression.The rMBP-S1 recombinant protein with good solubility and high expression was selected for further experiments.Western blot and indirect immunofluorescence assay(IFA)confirmed the correct expression and receptor binding of the protein.Aptamer Apt-S1-3 and Apt-S1-11 were enriched with the highest affinity(18%and 16.2%,respectively)and specificity,respectively.ELASA determined a dissociation constant of 2.09 nmol for Apt-S1-3,which showed no cross-reactivity with rMBP,rMBP-gD,rMBP-GP5,or BSA proteins.Mfold analysis and molecular docking simulations revealed the aptamer’s ability to form stable complexes with the target protein.IFA and flow cytometry confirmed the aptamer’s recognition and inhibition of PEDV infection.This study successfully screened a DNA aptamer that binds to the PEDV S1-CTD protein with high affinity and specificity,inhibiting virus infection and providing a foundation for targeted therapy and detection methods for PEDV.

关 键 词：猪流行性腹泻病毒 S1-CTD蛋白 磁珠法 核酸适配体 亲和力 

分 类 号：S852.659.6[农业科学—基础兽医学]