猪可溶性CD40L三聚体蛋白的原核表达及其对口蹄疫病毒样颗粒疫苗免疫效果评价

doi:10.11843/j.issn.0366-6964.2025.10.027

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (10): 5072-5083.doi: 10.11843/j.issn.0366-6964.2025.10.027

猪可溶性CD40L三聚体蛋白的原核表达及其对口蹄疫病毒样颗粒疫苗免疫效果评价

贺艳艳¹^,²(), 陈凌波¹, 魏甜¹, 滕志东¹, 张韵¹, 张铭洋¹, 马文倩¹, 敬晓棋², 郭慧琛¹, 穆素雨¹^,*(), 孙世琪¹^,*()

1. 中国农业科学院兰州兽医研究所兰州大学动物医学与生物安全学院动物疫病防控全国重点实验室, 兰州 730030
2. 榆林学院现代农学院, 榆林 719000

收稿日期:2025-01-10 出版日期:2025-10-23 发布日期:2025-11-01
通讯作者: 穆素雨,孙世琪 E-mail:160631841@qq.com;mutou6633937@126.com;sunshiqi@caas.cn
作者简介:贺艳艳(2000-)，女，陕西榆林人，硕士生，主要从事动物病毒病研究，E-mail：160631841@qq.com
基金资助:
甘肃省科技重大专项(24ZDWA004)；宁夏重点研发计划(2024BBF02017)；中国博士后面上项目(2024M763620)

Prokaryotic Expression of Porcine Soluble CD40L Trimer Protein and Evaluation of Its Immune Effect against Foot-and-Mouth Disease Virus-Like Particles Vaccine

HE Yanyan¹^,²(), CHEN Lingbo¹, WEI Tian¹, TENG Zhidong¹, ZHANG Yun¹, ZHANG Mingyang¹, MA Wenqian¹, JING Xiaoqi², GUO Huichen¹, MU Suyu¹^,*(), SUN Shiqi¹^,*()

1. National Key Laboratory of Animal Disease Prevention and Control, School of Animal Medicine and Biosafety, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou University, Lanzhou 730030, China
2. College of Advanced Agricultural Sciences, Yulin University, Yulin 719000, China

Received:2025-01-10 Online:2025-10-23 Published:2025-11-01
Contact: MU Suyu, SUN Shiqi E-mail:160631841@qq.com;mutou6633937@126.com;sunshiqi@caas.cn

摘要/Abstract

摘要：

旨在评价猪可溶性CD40L三聚体蛋白对口蹄疫(foot-and-mouth disease，FMD)病毒样颗粒(virus-like particles，VLPs)疫苗的免疫增强效果。本研究将含猪CD40配体(CD40L)三聚体(5B CD40L)基因的质粒在大肠杆菌BL21(DE3)中表达，纯化的重组5B CD40L蛋白偶联FITC后与PBMCs进行反应，使用荧光显微镜研究CD40L蛋白与单核细胞反应的生物活性。进一步将5B CD40L、VLPs和5B CD40L-VLPs免疫豚鼠，通过检测中和抗体、脾淋巴细胞增殖以及实时RT-qPCR评估5B CD40L对VLPs疫苗免疫效果的影响。SDS-PAGE和Western blot结果显示，表达的重组5B CD40L蛋白大小约32 ku，确定最佳表达条件为16 ℃、OD_{600 nm}值为0.9、0.5 mmol L^-1浓度IPTG诱导12 h；流式细胞仪分析表明，重组CD40L蛋白能与猪外周血单个核细胞(peripheral blood mononuclear cells，PBMCs)表面的CD40蛋白分子结合。5B CD40L联合VLPs可以加速特异性抗体及中和抗体反应，提高脾淋巴细胞增殖水平，并且在5B CD40L-VLPs组中，脾脏细胞的IL-2和INF-γ mRNA显著高于VLPs组。综上所述，5B CD40L具有增强FMD VLPs疫苗体液免疫应答和细胞免疫应答的能力，具有作为疫苗佐剂的潜力。

关键词: 5B CD40L, 口蹄疫, 免疫效果

Abstract:

This study aimed to evaluate the immune-enhancing effect of porcine soluble CD40L trimer protein on the Virus-like particles (VLPs) vaccine of Foot-and-mouth disease (FMD). In this study, the plasmid containing porcine CD40 ligand (CD40L) trimer (5B CD40L) gene was expressed in Escherichia coli BL21(DE3). The purified recombinant 5B CD40L protein was coupled to FITC and reacted with PBMCs. The biological activity of CD40L protein reacting with monocytes was studied by fluorescence microscopy. The guinea pigs were further immunized with 5B CD40L, VLPs and 5B CD40L-VLPs, and the effects of 5B CD40L on the immune effect of VLPs vaccine were evaluated by neutralizing antibodies, splenic lymphocyte proliferation and real-time RT-qPCR. SDS-PAGE and Western blot results showed that the size of recombinant 5B CD40L protein expressed was about 32 ku. The optimal expression conditions were 16℃, OD_{600 nm} value was 0.9, and concentration was 0.5 mmol L^-1 after IPTG induction for 12 h. Flow cytometry analysis showed that the recombinant CD40L protein could bind to CD40 protein molecules on the surface of porcine peripheral blood mononuclear cells (PBMCs). The combination of 5B CD40L and VLPs can accelerate the reaction of specific antibody and neutralizing antibody, and improve the proliferation level of spleen lymphocytes. In the 5B CD40L-VLPs group, the IL-2 and INF-γ mRNA of spleen cells are significantly higher than those in the VLPs group. In summary, 5B CD40L has the ability to enhance humoral and cellular immune responses of FMD VLPs vaccine, and has the potential as a vaccine adjuvant.

Key words: 5B CD40L, Foot-and-mouth disease, immune effect

中图分类号:

S855

贺艳艳, 陈凌波, 魏甜, 滕志东, 张韵, 张铭洋, 马文倩, 敬晓棋, 郭慧琛, 穆素雨, 孙世琪. 猪可溶性CD40L三聚体蛋白的原核表达及其对口蹄疫病毒样颗粒疫苗免疫效果评价[J]. 畜牧兽医学报, 2025, 56(10): 5072-5083.

HE Yanyan, CHEN Lingbo, WEI Tian, TENG Zhidong, ZHANG Yun, ZHANG Mingyang, MA Wenqian, JING Xiaoqi, GUO Huichen, MU Suyu, SUN Shiqi. Prokaryotic Expression of Porcine Soluble CD40L Trimer Protein and Evaluation of Its Immune Effect against Foot-and-Mouth Disease Virus-Like Particles Vaccine[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 5072-5083.

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参考文献 31

1	KARPUSASM,HSUY M,WANGJ H,et al.2 A crystal structure of an extracellular fragment of human CD40 ligand[J].Structure,1995,3(10):1031-1039. doi: 10.1016/S0969-2126(01)00239-8
2	ELGUETAR,BENSONM J,DE VRIESV C,et al.Molecular mechanism and function of CD40/CD40L engagement in the immune system[J].Immunol Rev,2009,229(1):152-172. doi: 10.1111/j.1600-065X.2009.00782.x
3	TAKADAY K,SHIMODAM,MAVERAKISE,et al.Soluble CD40L activates soluble and cell-surface integrin αvβ3, α5β1, and α4β1 by binding to the allosteric ligand-binding site (site 2)[J].J Biol Chem,2021,296,100399. doi: 10.1016/j.jbc.2021.100399
4	JOHNSONS,ZHANY,SUTHERLANDR M,et al.Selected Toll-like receptor ligands and viruses promote helper-independent cytotoxic T cell priming by upregulating CD40L on dendritic cells[J].Immunity,2009,30(2):218-227. doi: 10.1016/j.immuni.2008.11.015
5	CLARKL B,FOYT M,NOELLER J.CD40 and its ligand[J].Adv Immunol,1996,63,43-78.
6	SCHNABELCL,FLETEMEYERB,LVBKES,et al.CD154 Expression indicates T Cell activation following tetanus toxoid vaccination of horses[J].Front Immunol,2022,13,805026. doi: 10.3389/fimmu.2022.805026
7	VAN KOOTENC,BANCHEREAUJ.CD40-CD40 ligand[J].J Leukoc Biol,2000,67(1):2-17. doi: 10.1002/jlb.67.1.2
8	GRAFD,MVLLERS,KORTHÄUERU,et al.A soluble form of TRAP (CD40 ligand) is rapidly released after T cell activation[J].Eur J Immunol,1995,25(6):1749-1754. doi: 10.1002/eji.1830250639
9	CONLEYM E,DOBBSA K,FARMERD M,et al.Primary B cell immunodeficiencies: comparisons and contrasts[J].Annu Rev Immunol,2009,27,199-227. doi: 10.1146/annurev.immunol.021908.132649
10	BISHOPG A,MOOREC R,XIEP,et al.TRAF proteins in CD40 signaling[J].Adv Exp Med Biol,2007,597,131-151.
11	HASSANG S,STAGGJ,MOURADW.Role of CD154 in cancer pathogenesis and immunotherapy[J].Cancer Treat Rev,2015,41(5):431-440. doi: 10.1016/j.ctrv.2015.03.007
12	ZACCARDCR,WATKINSSC,KALINSKIP,et al.CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type 1 immunity[J].J Immunol,2015,194(3):1047-1056. doi: 10.4049/jimmunol.1401832
13	GUPTAS,TERMINIJ M,RIVASY,et al.A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model[J].Vaccine,2015,33(38):4798-4806. doi: 10.1016/j.vaccine.2015.07.081
14	SUNL,LIY,ZHANGY,et al.Adaptation and attenuation of duck Tembusu virus strain Du/CH/LSD/110128 following serial passage in chicken embryos[J].Clin Vaccine Immunol,2014,21(8):1046-1053. doi: 10.1128/CVI.00154-14
15	KWAS,SADAGOPALS,SHENX,et al.CD40L-adjuvanted DNA/modified vaccinia virus Ankara simian immunodeficiency virus (SIV) vaccine enhances protection against neutralization-resistant mucosal SIV infection[J].J Virol,2015,89(8):4690-4695. doi: 10.1128/JVI.03527-14
16	LIUS A,HAQUEM,STANFIELDB,et al.A recombinant fusion protein consisting of West Nile virus envelope domain Ⅲ fused in-frame with equine CD40 ligand induces antiviral immune responses in horses[J].Vet Microbiol,2017,198,51-58. doi: 10.1016/j.vetmic.2016.12.008
17	贺艳艳,郭慧琛,孙世琪,等.猪CD40L的可溶表达及其生物学活性检测[J].中国兽医科学,2025,55(3):389-395.
	HEY Y,GUOH C,SUNS Q,et al.Soluble expression and biological activity detection of CD40L in porcine[J].Chinese Journal of Veterinary Sciences,2025,55(3):389-395.
18	IDRISSH T,NAISMITHJ H.TNF alpha and the TNF receptor superfamily: structure-function relationship(s)[J].Microsc Res Tech,2000,50(3):184-195. doi: 10.1002/1097-0029(20000801)50:3<184::AID-JEMT2>3.0.CO;2-H
19	MORRISA E,REMMELER L Jr,KLINKER,et al.Incorporation of an isoleucine zipper motif enhances the biological activity of soluble CD40L (CD154)[J].J Biol Chem,1999,274(1):418-423. doi: 10.1074/jbc.274.1.418
20	任丽华,罗新,董虎,等.CpG和poly Ⅰ: C增强口蹄疫病毒样颗粒疫苗免疫效果的研究[J].中国兽医科学,2024,54(07):859-865.
	RENL H,LUOX,DONGH,et al.Research on the enhancement of the immune effect of foot-and-mouth disease virus-like particle vaccine by CpG and poly Ⅰ: C[J].Chinese Journal of Veterinary Sciences,2024,54(07):859-865.
21	SAADATM,GANDOMKARM,BAHREINIPOURA,et al.Evaluation of the designed multi-epitope protein of Brucella melitensis in guinea pigs[J].Iran J Basic Med Sci,2021,24(6):833-841.
22	NAGASHIMAH,AOKAY,SAKOMURAY,et al.Matrix metalloproteinase 2 is suppressed by rapidil, a CD40-CD40 ligand pathway inhibitor, in human abdominal aortic aneurysm wall[J].J Vasc Surg,2004,39(2):447-53. doi: 10.1016/j.jvs.2003.07.005
23	BROUWERP J M,ANTANASIJEVICA,RONKA J,et al.Lassa virus glycoprotein nanoparticles elicit neutralizing antibody responses and protection[J].Cell Host Microbe,2022,30(12):1759-1772. doi: 10.1016/j.chom.2022.10.018
24	CYSTERJ G,ALLENC D C.B cell responses: Cell interaction dynamics and decisions[J].Cell,2019,177(3):524-540. doi: 10.1016/j.cell.2019.03.016
25	WOOLAVERR A,WANGX,DOLLINY,et al.TRAF2 deficiency in B cells impairs CD40-induced isotype switching that can be rescued by restoring NF-κB1 activation[J].J Immunol,2018,201(11):3421-3430. doi: 10.4049/jimmunol.1800337
26	BANCZYKD,KALIESK,NACHBARL,et al.Activated CD4⁺ T cells enter the splenic T-cell zone and induce autoantibody-producing germinal centers through bystander activation[J].Eur J Immunol,2014,44(1):93-102. doi: 10.1002/eji.201343811
27	KORNUTAC A,LANGELLOTTIC A,BIDARTJ E,et al.A plasmid encoding the extracellular domain of CD40 ligand and Montanide^TM GEL01 as adjuvants enhance the immunogenicity and the protection induced by a DNA vaccine against BoHV-1[J].Vaccine,2021,39(6):1007-1017. doi: 10.1016/j.vaccine.2020.11.071
28	PUJOLJ,BOUILLENNEF,FARNIRF,et al.Generation of a soluble recombinant trimeric form of bovine CD40L and its potential use as a vaccine adjuvant in cows[J].Vet Immunol Immunopathol,2015,168(1-2):1-13. doi: 10.1016/j.vetimm.2015.09.011
29	SÁNCHEZ RAMOSO,GONZÁLEZ POSEA,GÓMEZ-PUERTAS,et al.Avian CD154 enhances humoral and cellular immune responses induced by an adenovirus vector-based vaccine in chickens[J].Comp Immunol Microbiol Infect Dis,2011,34(3):259-265. doi: 10.1016/j.cimid.2010.12.001
30	KWAS,LAIL,GANGADHARAS,et al.CD40L-adjuvanted DNA/modified vaccinia virus Ankara simian immunodeficiency virus SIV239 vaccine enhances SIV-specific humoral and cellular immunity and improves protection against a heterologous SIVE660 mucosal challenge[J].J Virol,2014,88(17):9579-9589. doi: 10.1128/JVI.00975-14
31	ROUSSETF,GARCIAE,BANCHEREAUJ.Cytokine-induced proliferation and immunoglobulin production of human B lymphocytes triggered through their CD40 antigen[J].J Exp Med,1991,173(3):705-710. doi: 10.1084/jem.173.3.705

引物名称 Primer name	引物序列(5′→3′) Primer sequence
IL-2 F	GTTATGCTTTCTCAGAGCAACCC
IL-2 R	GCTAAATTTAGCACCTGCTCCAC
IL-4 F	TGACGGTCATTCTCTTCTGCCTC
IL-4 R	AGGAGAGTGTGTTGAGGTGCTG
IL-12 F	ACCTCCCTAGGGCCTCACCAG
IL-12 R	GGCCTTGTGAAGTTCACTGTTC
IFN-γ F	ATGTTGGCTCTTCAGTTCTG
IFN-γ R	CATCTGAGTTATCTGCATTC
GAPDHF	CGAGACAAGATGGTGAAGGTC
GAPDHR	CATTGATGGCTACAATATCCACT

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猪可溶性CD40L三聚体蛋白的原核表达及其对口蹄疫病毒样颗粒疫苗免疫效果评价

Prokaryotic Expression of Porcine Soluble CD40L Trimer Protein and Evaluation of Its Immune Effect against Foot-and-Mouth Disease Virus-Like Particles Vaccine

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动物分组 Animal group	数量 Number	VLPs剂量/μg VLPs dosage	5B CD40L剂量/μg 5B CD40L dosage
5B CD40L	6	0	100
VLPs	6	100	0
5B CD40L+VLPs	6	100	100