携带猪流行性腹泻病毒抗原表位的铁蛋白纳米颗粒制备与免疫原性分析

doi:10.16431/j.cnki.1671-7236.2023.04.028

Abstract

Abstract: 【Objective】 The purpose of this experiment was to prepare ferritin nanoparticles carrying the epitope of Porcine epidemic diarrhea virus (PEDV) antigen and evaluate their immunogenicity,so as to provide a new candidate subunit vaccine for the prevention and control of PEDV.【Method】 The COE and S1D regions of PEDV fibrin S were designed as the tandem epitope S1CD,and the codon was optimized and synthesized.The recombinant plasmid pET28a-S1CD expressing S1CD protein alone and the recombinant plasmid pET28a-S1CD-Ferritin fusion expression S1CD protein and ferritin subunit were constructed,and then induced in E.coli and purified.SDS-PAGE and Western blotting were used to verify the expression of S1CD and S1CD-Ferritin proteins in E.coli.The morphology and structure of S1CD and S1CD-Ferritin proteins were observed by transmission electron microscope.The mice were immunized with purified S1CD-Ferritin protein,and S1CD protein and PBS were used as controls.Indirect ELISA was used to detect the level of S-specific antibody and the content of interferon-γ (IFN-γ).The level of neutralizing antibody was determined by neutralization test,and the immunogenicity of recombinant protein in mice was analyzed.【Result】 The recombinant plasmids pET28a-S1CD and pET28a-S1CD-Ferritin were successfully constructed.The result of SDS-PAGE showed that the recombinant protein S1CD and S1CD-Ferritin were efficiently expressed,with the size of about 22 and 39 ku,respectively,and both existed in the form of inclusion bodies.Western blotting results showed that the purified proteins could react specifically with the PEDV positive serum,indicating that the recombinant proteins S1CD and S1CD-Ferritin expressed by prokaryotic expression system had good reactivity.The result of transmission electron microscope showed that the purified S1CD-Ferritin protein could self-assemble into uniform nanoparticles with the diameter of 12-20 nm,indicating that S1CD-Ferritin protein had been successfully assembled into nanoparticles.Serums antibody test results showed that 7 days after the first immunization,the titer of PEDV specific antibody in S1CD-Ferritin group was significantly higher than that in S1CD group (P＜0.05).28 days after the first immunization,the level of neutralizing antibody and the content of IFN-γ were significantly higher than that in S1CD group (P＜0.05).【Conclusion】 The PEDV epitope fused with ferritin was highly expressed in E.coli,and could self-assemble into nanoparticles,and effectively enhance the immunogenicity of the protein antigens,which broadened the research ideas for studying new PEDV subunit vaccine.

Key words: Porcine epidemic diarrhea virus (PEDV); ferritin; nanoparticles; prokaryotic expression; immunogenicity

CLC Number:

S852.4

LIU Jiaxing, HAN Xueying, ZHANG Xinru, DENG Jiaxin, YAO Lunguang, LIU Yangkun. Preparation and Immunogenicity Analysis of Ferritin Nanoparticles Carrying Antigenic Epitopes of Porcine Epidemic Diarrhea Virus[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1567-1574.

References

[1] LEE C.Porcine epidemic diarrhea virus:An emerging and re-emerging epizootic swine virus[J].Virology Journal,2015,12(1):193.
[2] LIN F,ZHANG H,LI L,et al.PEDV:Insights and advances into types,function,structure,and receptor recognition[J].Viruses,2022,14(8):1744.
[3] WANG D,FANG L R,XIAO S B.Porcine epidemic diarrhea in China[J]. Virus Research,2016,226:7-13.
[4] LI Z,MA Z,LI Y,et al.Porcine epidemic diarrhea virus:Molecular mechanisms of attenuation and vaccines[J].Microbial Pathogenesis,2020,149:104553.
[5] JUNG K,SAIF L J.Porcine epidemic diarrhea virus infection:Etiology,epidemiology,pathogenesis and immunoprophylaxis[J].Veterinary Journal,2015,204(2):134-143.
[6] KOCHERHANS R,BRIDGEN A,ACKERMANN M,et al.Completion of the Porcine epidemic diarrhea coronavirus (PEDV) genome sequence[J].Virus Genes,2001,23(2):137-144.
[7] LIU C,TANG C,MA Y,et al.Receptor usage and cell entry of Porcine epidemic diarrhea coronavirus[J].Journal of Virology,2015,89(11):6121-6125.
[8] CHANG S H,BAE J L,KANG T J,et al.Identification of the epitope region capable of inducing neutralizing antibodies against the Porcine epidemic diarrhea virus[J].Molecules & Cells,2002,14(2):295-299.
[9] 李洁森,孙荣航,邝燕齐,等.猪流行性腹泻病毒S1D蛋白的优化表达及其单克隆抗体的制备[J].中国畜牧兽医,2021,48(12):4641-4651. LI J S,SUN R H,KUANG Y Q,et al.Optimal expression of Porcine epidemic diarrhea virus S1D protein and preparation of its monoclonal antibody[J].China Animal Husbandry & Veterinary Medicine,2021,48(12):4641-4651.(in Chinese)
[10] PAN J,CUI Z.Self-assembled nanoparticles:Exciting platforms for vaccination[J].Biotechnology Journal,2020,15(12):e2000087.
[11] MA H,LI X,LI J,et al.Immunization with a recombinant fusion of Porcine reproductive and respiratory syndrome virus modified GP5 and ferritin elicits enhanced protective immunity in pigs[J].Virology,2021,552:112-120.
[12] CHEN Y,HU Y,CHEN H,et al.A ferritin nanoparticle vaccine for Foot-and-mouth disease virus elicited partial protection in mice[J].Vaccine,2020,38(35):5647-5652.
[13] KANG Y F,SUN C,ZHUANG Z,et al.Rapid development of SARS CoV-2 spike protein receptor-binding domain self-assembled nanoparticle vaccine candidates[J].ACS Nano,2021,15(2):2738-2752.
[14] LIM J,CHEONG Y,KIM Y S,et al.RNA-dependent assembly of chimeric antigen nanoparticles as an efficient H5N1 pre-pandemic vaccine platform[J].Nanomedicine,2021,37:102438.
[15] 岳凯,韩雪英,冷超粮,等.猪重链铁蛋白的可溶性表达与纯化及纳米颗粒自组装[J].中国病原生物学杂志,2022,17(8):901-906. YUE K,HAN X Y,LENG C L,et al.Soluble expression and purification of Sus scrofa heavy chain ferritin and nanoparticles self-assembly[J].Journal of Pathogen Biology,2022,17(8):901-906.(in Chinese)
[16] WON H,LIM J,NOH Y H,et al.Efficacy of porcine epidemic diarrhea vaccines:A systematic review and meta-analysis[J].Vaccines (Basel),2020,8(4):642.
[17] ZHENG D,WANG X,JU N,et al.Immune responses in pregnant sows induced by recombinant Lactobacillus johnsonii expressing the COE protein of Porcine epidemic diarrhea virus provide protection for piglets against PEDV infection[J].Viruses,2021,14(1):7.
[18] MASUDA A,LEE J M,MIYATA T,et al.Stable trimer formation of spike protein from Porcine epidemic diarrhea virus improves the efficiency of secretory production in silkworms and induces neutralizing antibodies in mice[J].Veterinary Research,2021,52(1):102.
[19] GUO M,YI S,GUO Y,et al.Construction of a recombinant Lactococcus lactis strain expressing a variant Porcine epidemic diarrhea virus S1 gene and its immunogenicity analysis in mice[J].Viral Immunology,2019,32(3):144-150.
[20] XIANG S D,SCHOLZEN A,MINIGO G,et al. Pathogen recognition and development of particulate vaccines:Does size matter?[J].Methods,2006,40(1):1-9.
[21] MA X C,ZOU F,YU F,et al.Nanoparticle vaccines based on the receptor binding domain (RBD) and heptad repeat (HR) of SARS-CoV-2 elicit robust protective immune responses[J].Immunity,2020,53(6):1315-1330.
[22] KANEKIYO M,WEI C J,YASSINE H M,et al.Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies[J].Nature,2013,499(7456):102-106.
[23] YUAN C,ZHANG E,HUANG L,et al.Oral administration of inactivated Porcine epidemic diarrhea virus activate DCs in porcine Peyer's patches[J].BMC Veterinary Research,2018,14(1):239.
[24] LI Z,CUI K,WANG H,et al.A milk-based self-assemble Rotavirus VP6-ferritin nanoparticle vaccine elicited protection against the viral infection[J].Journal of Nanobiotechnology,2019,17(1):13.

Preparation and Immunogenicity Analysis of Ferritin Nanoparticles Carrying Antigenic Epitopes of Porcine Epidemic Diarrhea Virus

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	JIAO Linlin, CHENG Yuting, WU Qingguo, WU Shuang, WU Zhi, ZHU Shanyuan, QIAN Yingjuan. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2395-2402.
[2]	LI Can, LI Chuchu, ZENG Wei, ZHANG Ning, JI Chunxiao, CHEN Tao. Recombinant Expression and Function Study of RegⅢγ Protein in Porcine [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2414-2426.
[3]	SONG Yue, SU Shengjie, ZHANG Fan, BAI Fan, DAI Lingli, WANG Na, WANG Dawei, YANG Qianwen, ZHAO Shihua, ZHANG Yuemei. Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2479-2486.
[4]	ZHANG Fangyuan, LIN Yating, YANG Dawei, CHEN Hu, LI Guimei, SHAN Hu. Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2012-2022.
[5]	ZHANG Zhewei, DAI Xiaotong, WEI Zudan, CHEN Yunpeng, GAN Jing, BEI Weicheng. Study on Prokaryotic Expression and Immunogenicity of OmpW and TbpA Proteins in Swine Pasteurella multocida [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1489-1498.
[6]	HU Leyu, HU Xinyao, LI Ying, ZHAO Shengmiao, SHEN Fengzhen, WANG Changfa, LI Liangliang, WANG Tongtong, REN Huiying. Bioinformatics Analysis of HO-1 Gene and Preparation of Polyclonal Antibody in Dezhou Donkey [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1499-1510.
[7]	WANG Wenjie, RU Penghui, YU Chuan, DU Fuxi, CHEN Songbiao, SHANG Ke, ZHANG Chunjie, CHENG Xiangchao. Prokaryotic Expression of VP2 Protein of Luoyang Feline Panleukopenia Virus Isolate and Polyclonal Antibody Preparation [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1511-1521.
[8]	HUO Mingkai, GUAN Feihu, QIU Runhui, WEI Chunyan, YU Haitao, ZHU Jiale, ZHANG Hui. Study on the Mechanism of DDX1 Inhibiting Porcine Epidemic Diarrhea Virus Replication [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1556-1566.
[9]	JIA Xinyue, MA Jing, ZHANG Yanyan, MA Xun, WANG Zhengrong, BO Xinwen. Bioinformatics Analysis and Prokaryotic Expression of EGR-07243 and EGR-07243 Genes of Echinococcus granulosus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 847-858.
[10]	ZHANG Zhitao, LI Xianglong, KONG Lingli, LUO Yuxin, WANG Dongying. Prokaryotic Expression of Serine/Threonine Protein Phosphatase 2A in Fasciola gigantica and Expression Analysis at Different Stages [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1107-1117.
[11]	GAN Lu, ZHENG Huizhen, NUO Mingdalai, LIU Yan, JIN Min, HE Wenwen, WEN Licui, LI Yongchang, GAILIKE·Bayinchahan. Propagation of Trypanosoma evansi Yili Strain and Cloning, Expression and Bioinformatics Analysis of Its PFR Gene [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 469-478.
[12]	LYU Shuangfei, MA Xun, WANG Jing, KONG Cuilian, KOU Lijun, LIU Caixia, SHI Weidi, KANG Lichao, REN Huijie. Prokaryotic Expression and Immunoreactivity of PA-LF1 Fusion Protein of Bacillus anthracis [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 674-683.
[13]	ZHANG Kai, ZHANG Chunping, GE Shengqiang, SUN Chunxi, CHENG Jie, FU Chunyu, WANG Gang, NIU Xing, PENG Jun. Expression of p22 Protein of African Swine Fever Virus,Preparation of Polyclonal Antibody and Establishment of Indirect ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 270-279.
[14]	ZHU Lilin, WANG Houkun, CHEN Xueyang, LIU Jing, LIANG Xiongyan, FANG Chun, YANG Yuying. Effect of Chicken IFIT5 on Replication of J Subgroup Avian Leukemia Virus Within DF-1 Cells [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 310-316.
[15]	WANG Yuchen, TIAN Guangyuan, ZHOU Yaping, GUO Ting, ZHAO Hongmei, SUN Yajie, ZHAO Fengmiao, BIAN Yuchen, YU Jialiang, HAO Yongqing. Prokaryotic Expression of BPIV3 NP Protein and Its Immunoenhancing Effect on the BPIV3 Inactivated Vaccine [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3122-3130.