猪流行性腹泻病毒N蛋白原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2024.10.032

摘要/Abstract

摘要： 【目的】利用原核表达系统表达猪流行性腹泻病毒(Porcine epidemic diarrhea virus，PEDV)核衣壳蛋白(N)，并制备具有高效价和特异性强的多克隆抗体。【方法】利用生物信息学工具分析PEDV N蛋白氨基酸序列，并预测其抗原性；利用原核表达载体pColdⅠ诱导表达重组N蛋白，并通过SDS-PAGE和Western blotting鉴定重组蛋白；使用兔多抗制备佐剂与纯化后的重组N蛋白混合后免疫新西兰白兔，收集血清制备多克隆抗体。利用间接ELISA法测定重组N蛋白多克隆抗体效价，并对多克隆抗体进行Western blotting和间接免疫荧光试验(IFA)验证。【结果】生物信息学预测结果显示，N蛋白不含信号肽和跨膜结构，具有良好的抗原性和溶解度。SDS-PAGE电泳结果显示，重组N蛋白大小约为58 ku，主要以可溶性蛋白存在；Western blotting结果显示，该蛋白能与抗His标签的鼠源单克隆抗体发生特异性反应。间接ELISA结果显示，多克隆抗体效价可达1∶204 800；Western blotting结果表明，抗体稀释度为1∶5 000时能特异性识别重组N蛋白及PEDV感染后的Vero细胞样品中的N蛋白；IFA结果显示，当稀释度为1∶1 000时，该抗体能有效识别PEDV感染细胞样品中的N蛋白。【结论】本研究成功制备了效价高和特异性好的兔抗N蛋白多克隆抗体，为深入研究PEDV N蛋白的功能、了解PEDV复制机制和病毒-宿主细胞间的互作提供试验材料，为开发PEDV诊断和检测方法奠定基础。

关键词: 猪流行性腹泻病毒(PEDV); N蛋白; 原核表达; 多克隆抗体

Abstract: 【Objective】 The purpose of this study was to express the N protein of Porcine epidemic diarrhea virus (PEDV) using a prokaryotic expression system，and prepare polyclonal antibody with high efficiency and specificity.【Method】 Bioinformatics tools were used to analyze the amino acid sequence of PEDV N protein and predict its antigenicity. Recombinant N protein was induced by prokaryotic expression vector pColdⅠ，and the recombinant protein was identified by SDS-PAGE and Western blotting. New Zealand White rabbits were immunized with the preparation adjuvant of rabbit polyclonal antibody mixed with purified recombinant N protein，and serum was collected to prepare polyclonal antibody. The titer of recombinant N protein polyclonal antibody was determined by indirect ELISA，and the polyclonal antibody was verified by Western blotting and indirect immunofluorescence assay (IFA).【Result】 The results of bioinformatics prediction show that N protein didn’t contain signal peptide and transmembrane structure，and had good antigenicity and solubility. SDS-PAGE results showed that the recombinant N protein approximately 58 ku in size，existed primarily in a soluble form. Western blotting revealed that the protein specifically react with anti-His labeled mouse monoclonal antibodies . Indirect ELISA results demonstrated that the titer of polyclonal antibody could reach 1∶ 204 800. Western blotting results showed that recombinant N protein and N protein in PEDV infected Vero cell samples could be specifically identified when the antibody dilution was 1∶5 000. IFA results showed that when the dilution was 1∶1 000，the antibody could effectively identify N protein in PEDV-infected cell samples.【Conclusion】 The rabbit anti-N protein polyclonal antibody with high efficiency and good specificity was successfully prepared，providing experimental materials for further study of PEDV N protein function，understanding PEDV replication mechanism and virus-host cell interaction，and laying a foundation for the development of PEDV diagnosis and detection methods.

Key words: Porcine epidemic diarrhea virus (PEDV); N protein; prokaryotic expression; polyclonal antibody

中图分类号:

S852.65

吴婕, 杜菁, 樊繁, 任金阳, 卢会鹏, 张力, 雷昕诺, 曹世诺, 吴植, 朱睿, 朱善元. 猪流行性腹泻病毒N蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2024, 51(10): 4522-4530.

WU Jie, DU Jing, FAN Fan, REN Jinyang, LU Huipeng, ZHANG Li, LEI Xinnuo, CAO Shinuo, WU Zhi, ZHU Rui, ZHU Shanyuan. Prokaryotic Expression of N Protein of Porcine Epidemic Diarrhea Virus and Preparation of Polyclonal Antibodies[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(10): 4522-4530.

参考文献

[1] WANG L，BYRUM B，ZHANG Y. New variant of Porcine epidemic diarrhea virus，United States，2014[J]. Emerging Infectious Diseases，2014,20(5):917-919.
[2] JUNG K，SAIF L J，WANG Q. Porcine epidemic diarrhea virus (PEDV): An update on etiology，transmission，pathogenesis，and prevention and control[J]. Virus Research，2020,286:198045.
[3] CHEN P，WANG K，HOU Y，et al. Genetic evolution analysis and pathogenicity assessment of Porcine epidemic diarrhea virus strains circulating in part of China during 2011-2017[J]. Infection Genetics and Evolution，2019,69:153-165.
[4] HOU Y，WANG Q. Emerging highly virulent Porcine epidemic diarrhea virus: Molecular mechanisms of attenuation and rational design of live attenuated vaccines[J]. International Journal of Molecular Sciences，2019,20(21):5478.
[5] LIN F，ZHANG H，LI L，et al. PEDV: Insights and advances into types，function，structure，and receptor recognition[J]. Viruses-Basel，2022,14(8):1744.
[6] SHI D，SHI H，SUN D，et al. Nucleocapsid interacts with NPM1 and protects it from proteolytic cleavage，enhancing cell survival，and is involved in PEDV growth[J]. Scientific Reports，2017,7:39700.
[7] TAN Y W，FANG S，FAN H，et al. Amino acid residues critical for RNA-binding in the N-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of Coronavirus in cultured cells[J]. Nucleic Acids Research，2006,34(17):4816-4825.
[8] SU M，SHI D，XING X，et al. Coronavirus Porcine epidemic diarrhea virus nucleocapsid protein interacts with p53 to induce cell cycle arrest in S-phase and promotes viral replication[J]. Journal of Virology，2021,95(16):e18721.
[9] ZHU Q，SU M，WEI S，et al. Up-regulated 60S ribosomal protein L18 in PEDV N protein-induced S-phase arrested host cells promotes viral replication[J]. Virus Research，2022,321:198916.
[10] DING Z，FANG L，JING H，et al. Porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between IRF3 and TBK1[J]. Journal of Virology，2014,88(16):8936-8945.
[11] ADAMS M J，CARSTENS E B. Ratification vote on taxonomic proposals to the international committee on taxonomy of viruses (2012)[J]. Archives of Virology，2012,157(7):1411-1422.
[12] WANG D，FANG L，XIAO S. Porcine epidemic diarrhea in China[J] Virus Research，2016,226:7-13.
[13] 刘孝珍，陈建飞，时洪艳，等. 2011年猪流行性腹泻病毒的遗传变异分析[J]. 中国预防兽医学报，2012,34(3):180-183.LIU X Z，CHEN J F，SHI H Y，et al. Genetic variation analysis of Porcine epidemic diarrhea virus isolated in 2011[J]. Chinese Journal of Preventive Veterinary Medicine，2012,34(3):180-183. (in Chinese)
[14] 尹宝英，朱小甫，郑红青，等. 猪流行性腹泻病毒逃逸宿主天然免疫研究进展[J]. 动物医学进展，2023,44(10):89-94.YIN B Y，ZHU X F，ZHENG H Q，et al. Progress on Porcine epidemic diarrhea virus escaping from host innate immunity[J]. Progress in Veterinary Medicine，2023,44(10):89-94. (in Chinese)
[15] LI C，LU H，GENG C，et al. Epidemic and evolutionary characteristics of Swine enteric viruses in South-Central China from 2018 to 2021[J]. Viruses-Basel，2022,14(7):1420.
[16] 董世娟，谢春芳，司伏生，等. 猪流行性腹泻病毒免疫及疫苗研制[J]. 生物工程学报，2021,37(8):2603-2613.DONG S J，XIE C F，SI F S，et al. Immunization against Porcine epidemic diarrhea virus and vaccine development[J]. Chinese Journal of Biotechnology，2021,37(8):2603-2613. (in Chinese)
[17] 卢思嘉，郑兰兰. 猪流行性腹泻病毒疫苗研究进展[J]. 中国畜牧兽医，2023,50(7):2931-2940.LU S J，ZHENG L L. Research progress on Porcine epidemic diarrhea virus vaccines[J]. China Animal Husbandry & Veterinary Medicine，2023,50(7):2931-2940. (in Chinese)
[18] 左媛媛，徐天刚，戈胜强，等. 我国猪流行性腹泻病毒流行现状及分子遗传演化特征[J]. 中国动物检疫，2022,39(9):9-17.ZUO Y Y，XU T G，GE S Q，et al. Prevalence and molecular genetic evolution of Porcine epidemic diarrhea virus in China[J]. China Animal Health Inspection，2022,39(9):9-17. (in Chinese)
[19] GUO J，FANG L，YE X，et al. Evolutionary and genotypic analyses of global Porcine epidemic diarrhea virus strains[J]. Transboundary and Emerging Diseases，2019,66(1):111-118.
[20] 韦学雷，梁青青，曹贝贝，等. 猪Delta冠状病毒、猪传染性胃肠炎病毒和猪流行性腹泻病毒多重RT-PCR检测方法的建立及应用[J]. 中国兽医学报，2018,38(1):11-16.WEI X L，LIANG Q Q，CAO B B，et al. Establishment and application of a multiplex RT-PCR assay for simultaneous detection of Porcine delta coronavirus，Porcine transmissible gastroenteritis virus and Porcine epidemic diarrhea virus[J]. Chinese Journal of Veterinary Science，2018,38(1):11-16. (in Chinese)
[21] 郭楠楠. PEDV细胞适应株全基因组序列测定与分析和抗N蛋白高免血清的制备[D].南昌：江西农业大学，2018.GUO N N. Whole-genome analysis of Vero cell-adapted Porcine epidemic diarrhea virus and preparation of hyperimmune antiserum against PEDV N-protein[D]. Nanchang：Jiangxi Agricultural University，2018. (in Chinese)
[22] HU Y，XIE X，YANG L，et al. A comprehensive view on the host factors and viral proteins associated with Porcine epidemic diarrhea virus infection[J]. Frontiers in Microbiology，2021,12:762358.
[23] WEBER J，PENG H，RADER C. From rabbit antibody repertoires to rabbit monoclonal antibodies[J]. Experimental and Molecular Medicine，2017,49(3):e305.
[24] NIU Z，ZHANG S，XU S，et al. Porcine epidemic diarrhea virus replication in human intestinal cells reveals potential susceptibility to cross-species infection[J]. Viruses-Basel，2023,15(4):956.
[25] SU M，CHEN Y，QI S，et al. A mini-review on cell cycle regulation of Coronavirus infection[J]. Frontiers in Veterinary Science，2020,7:586826.
[26] YUAN X，YAO Z，WU J，et al. G1 phase cell cycle arrest induced by SARS-CoV 3a protein via the cyclin D3/pRb pathway[J]. American Journal of Respiratory Cell and Molecular Biology，2007,37(1):9-19.
[27] LIWNAREE B，NARKPUK J，SUNGSUWAN S，et al. Growth enhancement of Porcine epidemic diarrhea virus (PEDV) in Vero E6 cells expressing PEDV nucleocapsid protein[J]. PLoS One，2019,14(3):e212632.