猪流行性腹泻病毒S1蛋白的真核表达及中和性单克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2025.10.030

摘要/Abstract

摘要： 【目的】制备猪流行性腹泻病毒(Porcine epidemic diarrhea virus，PEDV)S1重组蛋白及其中和性单克隆抗体,为PEDV诊断试剂和新型疫苗研发提供材料。【方法】构建可表达S1蛋白的重组质粒，转染HEK293F细胞，以镍亲和层析和凝胶过滤层析进行蛋白纯化。将PEDV CH/Hubei/2016毒株接种于Vero细胞进行培养增殖，对病毒液进行浓缩并利用凝胶过滤层析纯化PEDV，以SDS-PAGE及Western blotting对纯化的PEDV进行鉴定后免疫BALB/c小鼠以制备杂交瘤细胞；以间接ELISA及免疫过氧化物酶单层细胞试验(IPMA)筛选阳性杂交瘤细胞，并通过病毒中和试验筛选鉴定抗PEDV S1蛋白中和性单克隆抗体。【结果】SDS-PAGE和Western blotting结果显示，S1重组蛋白在HEK293F细胞培养上清中高效表达。获得TCID₅₀为1×10^-6.5/0.1 mL纯化的PEDV。筛选获得5株稳定分泌抗PEDV S1重组蛋白单克隆抗体的杂交瘤细胞株2E1E10、4A8C8、8G9B11、9H8H1和10C9A5，其腹水的IPMA和ELISA效价分别为6.4×10^-4至1.28×10^-5和6.4×10^-5至1.024×10^-7。病毒中和试验结果显示，单克隆抗体2E1E10和4A8C8对PEDV有显著中和活性，对DR13毒株的半数抑制浓度(IC₅₀)分别为1.212和1.203 μg/mL，对CHHB毒株的IC₅₀分别为0.948和1.712 μg/mL。【结论】本研究获得了具有良好抗原活性的PEDV S1重组蛋白和抗S1蛋白中和性单克隆抗体,为PEDV诊断试剂和疫苗研发奠定良好的研究基础。

关键词: 猪流行性腹泻病毒(PEDV); S1蛋白; 单克隆抗体; 中和活性; 广谱反应性

Abstract: 【Objective】 The aim of this study was to prepare the S1 recombinant protein of Porcine epidemic diarrhea virus (PEDV) and its neutralizing monoclonal antibodies (mAbs),and provide critical materials for the development of PEDV diagnostic reagents and novel vaccines.【Method】 A recombinant plasmid that expressed S1 protein was constructed and transfected into HEK293F cells.Nickel affinity chromatography and gel filtration chromatography were used to purify the expressed protein. PEDV CH/Hubei/2016 strain was cultured in Vero cells for propagation.The viral suspension was subsequently concentrated and purified via gel filtration chromatography.The purified PEDV was characterized by SDS-PAGE and Western blotting.BALB/c mice were then immunized with the purified antigen to generate hybridoma cells.Positive hybridoma cells were screened by indirect ELISA and immunoperoxidase monolayel assay (IPMA).The neutralizing monoclonal antibodies against PEDV S1 protein were screened and identified using virus neutralization assay.【Result】 SDS-PAGE and Western blotting analysis demonstrated that S1 recombinant protein was efficiently expressed in HEK293F cell culture supernatant.Purified PEDV with TCID₅₀ of 1 ×10^-6.5/0.1 mL was obtained.Five hybridoma cell lines stably secreting anti-S1 mAbs were obtained and the IPMA and ELISA titers of the ascitics ranged from 6.4×10^-4 to 1.28×10^-5 and 6.4×10^-5 to 1.024×10^-7,respectively.Virus neutralization assay revealed that mAbs 2E1E10 and 4A8C8 exhibited high neutralizing activity,of which the inhibitory concentration 50% (IC₅₀) values were calculated as 1.212 and 1.203 μg/mL for strain DR13,and 0.948 and 1.712 μg/mL for strain CHHB,respectively.【Conclusion】 PEDV S1 recombinant protein with high antigenic activity was obtained,and neutralizing monoclonal antibodies against S1 protein were produced,which providing a good research foundation for the development of PEDV diagnostic reagents and vaccines.

Key words: Porcine epidemic diarrhea virus (PEDV); S1 protein; monoclonal antibodies; neutralizing activity; broad-spectrum reactivity

中图分类号:

S852.65⁺9.6

李春震, 李青梅, 邹婉莹, 孟泽锟, 孙亚宁, 杨苏珍, 郭军庆, 张改平. 猪流行性腹泻病毒S1蛋白的真核表达及中和性单克隆抗体制备[J]. 中国畜牧兽医, 2025, 52(10): 4854-4863.

LI Chunzhen, LI Qingmei, ZOU Wanying, MENG Zekun, SUN Yaning, YANG Suzhen, GUO Junqing, ZHANG Gaiping. Eukaryotic Expression of S1 Protein of Porcine Epidemic Diarrhea Virus and Preparation of Its Neutralizing Monoclonal Antibody[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(10): 4854-4863.

参考文献

[1] 吴婕,杜菁,樊繁,等.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 J,DU J,FAN F,et al.Prokaryotic expression of N protein of Porcine epidemic diarrhea virus and preparation of polyclonal antibodies[J].China Animal Husbandry & Veterinary Medicine,2024,51(10):4522-4530.(in Chinese)
[2] PENSAERT M B,DE BOUCK P.A new Coronavirus-like particle associated with diarrhea in swine[J].Archives of Virology,1978,58(3):243.
[3] HANKE D,JENCKEL M,PETROV A,et al.Comparison of Porcine epidemic diarrhea viruses from Germany and the United States,2014[J]. Emerging Infectious Diseases,2015,21(3):493-496.
[4] OKA T,SAIF L J,MARTHALER D,et al.Cell culture isolation and sequence analysis of genetically diverse US Porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene[J].Veterinary Microbiology,2014,173(3-4):258-269.
[5] SUZUKI T,MURAKAMI S,TAKAHASHI O,et al.Molecular characterization of Pig epidemic diarrhoea viruses isolated in Japan from 2013 to 2014[J].Infection,Genetics and Evolution,2015,36:363-368.
[6] JI Z,SHI D,SHI H,et al.A Porcine epidemic diarrhea virus strain with distinct characteristics of four amino acid insertion in the COE region of spike protein[J].Veterinary Microbiology,2021,253:108955.
[7] SUN R Q,CAI R J,CHEN Y Q,et al.Outbreak of Porcine epidemic diarrhea in suckling piglets,China[J].Emerging Infectious Diseases,2012,18(1):161-163.
[8] CHEN Q,GAUGER P C,STAFNE M R,et al.Pathogenesis comparison between the united states Porcine epidemic diarrhoea virus prototype and S-INDEL-variant strains in conventional neonatal piglets[J].Journal of General Virology,2016,97(5):1107-1121.
[9] CHEN Y,HE Z,LUO Y,et al.Tris stabilized AuNPs based lateral flow immunochromatography for the simultaneous detection of Porcine epidemic diarrhea virus and Rotavirus on-site[J].Spectrochimica Acta.Part A,Molecular and Biomolecular Spectroscopy,2024,320:124670.
[10] GARCÍA-HERNÁNDEZ M E,TRUJILLO-ORTEGA M E,ALCARAZ-ESTRADA S L,et al.Molecular detection and characterization of Porcine epidemic diarrhea virus and Porcine aichivirus C coinfection in México[J].Viruses,2021,13(5):738.
[11] WANG Q,ZHANG Q,SHI X,et al.ACADM inhibits AMPK activation to modulate PEDV-induced lipophagy and β-oxidation for impairing viral replication[J].Journal of Biological Chemistry,2024,300(8):107549.
[12] 彭棋,张雪,赫文龙,等.猪流行性腹泻病毒G2a变异株CH-HK-2021的分离鉴定及其遗传进化分析[J].南方农业学报,2022,53(8):2077-2087. PENG Q,ZHANG X,HE W L,et al.Isolation，identification，and genetic evolution analysis of variant strain CH-HK-2021 of G2a Porcine epidemic diarrhea virus[J].Journal of Southern Agriculture,2022,53(8):2077-2087.(in Chinese)
[13] LIU J,SHI H,CHEN J,et al.A new neutralization epitope in the spike protein of Porcine epidemic diarrhea virus[J].International Journal of Molecular Sciences,2022,23(17):9674.
[14] THAVORASAK T,CHULANETRA M,GLAB-AMPAI K,et al.Novel neutralizing epitope of PEDV S1 protein identified by IgM monoclonal antibody[J]. Viruses,2022,14(1):125.
[15] GONG L,LIN Y,QIN J,et al.Neutralizing antibodies against Porcine epidemic diarrhea virus block virus attachment and internalization[J].Virology Journal,2018,15(1):133.
[16] SUN Y G,LI R,XIE S,et al.Identification of a novel linear B-cell epitope within the collagenase equivalent domain of Porcine epidemic diarrhea virus spike glycoprotein[J].Virus Research,2019,266:34-42.
[17] 常向彩,杨晓农,朱子凤,等.两种纯化兔抗酸性红73免疫球蛋白G方法的比较研究[J].中国畜牧兽医,2013,40(7):124-126. CHANG X C,YANG X N,ZHU Z F,et al.Comparison of two methods for purifying the rabbit antibody of acid red 73 IgG[J].China Animal Husbandry & Veterinary Medicine,2013,40(7):124-126.(in Chinese)
[18] LI M,WANG Y,WANG Y,et al.Accurate location of two conserved linear epitopes of PEDV utilizing monoclonal antibodies induced by S1 protein nanoparticles[J].International Journal of Biological Macromolecules,2023,253(6):127276.
[19] LI W,VAN KUPPEVELD F J M,HE Q,et al.Cellular entry of the Porcine epidemic diarrhea virus[J].Virus Research,2016,226:117-127.
[20] KIRCHDOERFER R N,BHANDARI M,MARTINI O,et al.Structure and immune recognition of the Porcine epidemic diarrhea virus spike protein[J]. Structure (London,England:1993),2021,29(4):385-392.e5.
[21] POLYIAM K,RUENGJITCHATCHAWALYA M,MEKVICHITSAENG P,et al.Immunodominant and neutralizing linear B-cell epitopes spanning the spike and membrane proteins of Porcine epidemic diarrhea virus[J].Frontiers in Immunology,2021,12:785293.
[22] 柏家果,刘思雨,杜琛,等.猪流行性腹泻病毒S蛋白的原核表达及其多克隆抗体的制备[J].中国兽医科学,2022,52(11):1415-1421. BAI J G,LIU S Y,DU C,et al.Prokaryotic expression and polyclonal antibody preparation of spike protein of Porcine epidemic diarrhea virus[J]. Chinese Veterinary Science,2022,52(11):1415-1421.(in Chinese)
[23] 柴茂,马震原,杨海波,等.基于猪流行性腹泻病毒S蛋白单克隆抗体的竞争ELISA方法的建立[J].中国兽医科学,2025,55(1):73-79. CHAI M,MA Z Y,YANG H B,et al.Establishment of a competitive ELISA method based on monoclonal antibodies against Porcine epidemic diarrhea virus S protein[J].Chinese Veterinary Science,2025,55(1):73-79.(in Chinese)
[24] LANGEL S N,PAIM F C,ALHAMO M A,et al.Stage of gestation at Porcine epidemic diarrhea virus infection of pregnant swine impacts maternal immunity and lactogenic immune protection of neonatal suckling piglets[J]. Frontiers in Immunology,2019,10:727.
[25] AMIMO J O,MICHAEL H,CHEPNGENO J,et al.Maternal immunization and vitamin A sufficiency impact sow primary adaptive immunity and passive protection to nursing piglets against Porcine epidemic diarrhea virus infection[J].Frontiers in Immunology,2024,15:1397118.
[26] LEE D H,JEON Y S,PARK C K,et al.Immunoprophylactic effect of chicken egg yolk antibody (IgY) against a recombinant S1 domain of the Porcine epidemic diarrhea virus spike protein in piglets[J].Archives of Virology,2015,160(9):2197-2207.
[27] ZHANG F,CHEN Y,KE Y,et al.Single chain fragment variable (scFv) antibodies targeting the spike protein of Porcine epidemic diarrhea virus provide protection against viral infection in piglets[J].Viruses,2019,11(1):58.
[28] SHI W,HAO H,LI M,et al.Expression and purification of a PEDV-neutralizing antibody and its functional verification[J].Viruses,2021,13(3):472.
[29] WANG S,WANG Z,LI Y,et al.Generation of whole-porcine neutralizing antibodies of an Alphacoronavirus by single B cell antibody technology[J].Antiviral Research,2023,220:105754.
[30] LI C,LI W,LUCIO DE ESESARTE E,et al.Cell attachment domains of the Porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies[J].Journal of Virology,2017,91(12):e00273-17.
[31] 马宇聪,刘增素,王书博,等.PEDV双抗体夹心ELISA检测方法的建立[J].中国兽医科学,2019,49(9):1152-1159. MA Y C,LIU Z S,WANG S B,et al.Establishment of a double antibody sandwich ELISA for the detection of PEDV[J].Chinese Veterinary Science,2019,49(9):1152-1159.(in Chinese)
[32] 王一丹,杨发龙,陈弟诗,等.猪腹泻病毒一步法多重TaqMan荧光定量RT-PCR检测法的建立及应用[J].中国农业科学,2023,56(1):179-192. WANG Y D,YANG F L,CHEN D S,et al.One-step multiple TaqMan Real-time RT-PCR for simultaneous detection of Swine diarrhea viruses[J] Scientia Agricultura Sinica,2023,56(1):179-192.(in Chinese)
[33] HOU W,FAN M,ZHU Z,et al.Establishment and application of a triplex Real-time RT-PCR assay for differentiation of PEDV,PoRV,and PDCoV[J].Viruses,2023,15(6):1238.