产气荚膜梭菌α毒素-铁蛋白纳米颗粒抗原制备及其对小鼠的免疫原性评价

doi:10.11843/j.issn.0366-6964.2025.09.040

Abstract

Abstract:

This study aimed to prepare Clostridium perfringens α-ferritin nanoparticle antigens and evaluate its immunogenicity in mice. The full-length α gene containing D56G and H68G point mutations and the Spytag sequence, as well as the gene fragment ferritin containing the ferritin gene and SpyCatcher sequence were cloned into the prokaryotic expression vector pET 28a vector respectively. Recombinant proteins were expressed followed by identification and purification. The cytotoxicity, lecithinase activity and haemolytic activity of purified α_m2ST were analysed. The purified α_m2ST and FeSC were docked in vitro. The docking product of α-ferritin (α_m2-Fe) and α_m2ST were mixed with adjuvant, respectively, and used for mice immunization. Mice serum was collected every week post immunization to measure IgG, IgG subtype antibody levels and the serum neutralization titers; on day 28 post the second immunization, splenocytes were isolated to analyze T-cell subsets and IFN-γ secretion levels. The results showed that at 28 ℃, α_m2ST and FeSC were predominantly expressed in a soluble manner. The α_m2ST was free of cytotoxicity, lecithinase activity, or hemolytic activity after detection, indicating that the α_m2ST is non-toxic. After in vitro docking of α_m2ST and FeSC, it was confirmed that the α_m2-Fe formed nanoparticles with a diameter of 32.7-50.7 nm, with a main peak at 37.8 nm, after analyses with SDS-PAGE, TEM, and DLS. Following immunization in mice, the α_m2-Fe group exhibited high levels of IgG2a antibodies (P < 0.001). On day 21 after the second immunization, the serum neutralization titer in the α_m2-Fe group was 64-fold, significantly surpassing the 32-fold titer in the α_m2ST group (P < 0.05). By day 28 after the second immunization, the IFN-γ levels in the supernatant of stimulated splenocytes were also higher in the α_m2-Fe group compared to the α_m2ST group (P < 0.05). Flow cytometry analysis revealed that the α_m2-Fe group produced a higher proportion of effector CD8⁺ T cells (P < 0.000 1). Mice experiments confirmed that the α_m2-Fe nanoparticle antigens effectively induce both humoral and cellular immunity in mice.

Key words: Clostridium perfringens, alpha toxin, ferritin, nanoparticles, immunogenicity

CLC Number:

S852.616.3

ZHAO Huiyu, LEI Yinuo, XING Qianru, ZHANG Shan, ZHANG Guangzhi, JIANG Hui, SHEN Qingchun, DING Jiabo, JIANG Shijin, FAN Xuezheng. Preparation of Clostridium perfringens α Toxin-ferritin Nanoparticle Antigens and Evaluation of Its Immunogenicity in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4626-4637.

Figures/Tables 10

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References 39

1	UZALF A,FREEDMANJ C,SHRESTHAA,et al.Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease[J].Future Microbiol,2014,9(3):361-377. doi: 10.2217/fmb.13.168
2	OHTANIK,SHIMIZUT.Regulation of toxin production in Clostridium perfringens[J].Toxins (Basel),2016,8(7):207. doi: 10.3390/toxins8070207
3	SUZAKIA,HAYAKAWAS.Clinical and microbiological features of fulminant haemolysis caused by Clostridium perfringens bacteraemia: unknown pathogenesis[J].Microorganisms,2023,11(4):824. doi: 10.3390/microorganisms11040824
4	SANTOSR,ABDEL-NOURJ,MCAULEYC,et al.Clostridium perfringens associated with dairy farm systems show diverse genotypes[J].Int J Food Microbiol,2022,382,109933. doi: 10.1016/j.ijfoodmicro.2022.109933
5	TIANR,XUS,LIP,et al.Characterization of G-type Clostridium perfringens bacteriophages and their disinfection effect on chicken meat[J].Anaerobe,2023,81,102736. doi: 10.1016/j.anaerobe.2023.102736
6	KAWAMURAT,PRAHI,MAHAZUS,et al.Types A and F Clostridium perfringens in healthcare wastewater from Ghana[J].Appl Environ Microbiol,2023,89(12):e0161923. doi: 10.1128/aem.01619-23
7	LEED,JANGG,MINK C,et al.Coinfection with porcine epidemic diarrhea virus and Clostridium perfringens type A enhances disease severity in weaned pigs[J].Arch Virol,2023,168(6):166. doi: 10.1007/s00705-023-05798-3
8	LUR,LIUB,WUL,et al.A broad-spectrum phage endolysin (LysCP28) able to remove biofilms and inactivate Clostridium perfringens strains[J].Foods,2023,12(2):411. doi: 10.3390/foods12020411
9	MCDONELJ L.Clostridium perfringens toxins (type A, B, C, D, E)[J].Pharmacol Ther,1980,10(3):617-655. doi: 10.1016/0163-7258(80)90031-5
10	ROODJ I,ADAMSV,LACEYJ,et al.Expansion of the Clostridium perfringens toxin-based typing scheme[J].Anaerobe,2018,53,5-10. doi: 10.1016/j.anaerobe.2018.04.011
11	OUL,YEB,SUNM,et al.Mechanisms of intestinal epithelial cell damage by Clostridium perfringens[J].Anaerobe,2024,87,102856. doi: 10.1016/j.anaerobe.2024.102856
12	MEHDIZADEH GOHARII,A NAVARROM,LIJ,et al.Pathogenicity and virulence of Clostridium perfringens[J].Virulence,2021,12(1):723-753. doi: 10.1080/21505594.2021.1886777
13	UZALF A,GIANNITTIF,ASINJ.Yellow lamb disease (Clostridium perfringens type A enterotoxemia of sheep): A Review[J].Animals (Basel),2022,12(12):1590.
14	JIANGY F,MAY H,LIUQ Q,et al.Tracing Clostridium perfringens strains from beef processing of slaughter house by pulsed-field gel electrophoresis, and the distribution and toxinotype of isolates in Shaanxi province, China[J].Food Microbiol,2022,101,103887. doi: 10.1016/j.fm.2021.103887
15	MOREIRAG M,SALVARANIF M,CUNHAC E,et al.Immunogenicity of a trivalent recombinant vaccine against Clostridium perfringens alpha, beta, and epsilon toxins in farm ruminants[J].Sci Rep,2016,6,22816. doi: 10.1038/srep22816
16	SMITHL A.Botulism and vaccines for its prevention[J].Vaccine,2009,27(4):D33-D39.
17	LANIGANT M,KOPERAH C,SAUNDERST L.Principles of genetic engineering[J].Genes (Basel),2020,11(3):291. doi: 10.3390/genes11030291
18	邬沛伶,李依璇,王浩杰,等.猪流行性腹泻疫苗研究进展[J].畜牧兽医学报,2025,56(3):1042-1058. doi: 10.11843/j.issn.0366-6964.2025.03.007
	WUP L,LIY X,WANGH J,et al.Research progress of porcine epidemic diarrhea vaccine for pigs[J].Acta Veterinaria et Zootechnica Sinica,2025,56(3):1042-1058. doi: 10.11843/j.issn.0366-6964.2025.03.007
19	EVERSM J W,VAN DE WAKKERS I,DE GROOTE M,et al.Functional siRNA delivery by extracellular vesicle-Liposome hybrid nanoparticles[J].Adv Healthc Mater,2022,11(5):e2101202. doi: 10.1002/adhm.202101202
20	HOJ K,JEEVAN-RAJB,NETTERH J.Hepatitis B virus (HBV) subviral particles as protective vaccines and vaccine platforms[J].Viruses,2020,12(2):126. doi: 10.3390/v12020126
21	YUJ,THOMASP V,SCIACCAM,et al.Ad26.COV2.S and SARS-CoV-2 spike protein ferritin nanoparticle vaccine protect against SARS-CoV-2 Omicron BA.5 challenge in macaques[J].Cell Rep Med,2023,4(4):101018. doi: 10.1016/j.xcrm.2023.101018
22	HANJ A,KANGY J,SHINC,et al.Ferritin protein cage nanoparticles as versatile antigen delivery nanoplatforms for dendritic cell(DC)-based vaccine development[J].Nanomedicine,2014,10(3):561-569. doi: 10.1016/j.nano.2013.11.003
23	李志鹏,刘福航,崔奎青,等.铁蛋白Ferritin原核表达和纯化及纳米颗粒胞外自组装[J].畜牧兽医学报,2018,49(1):75-82. doi: 10.11843/j.issn.0366-6964.2018.01.009
	LIZ P,LIUF H,CUIK Q,et al.Prokaryotic expression and purification of ferritin and nano-particles self-assembling in vitro[J].Acta Veterinaria et Zootechnica Sinica,2018,49(1):75-82. doi: 10.11843/j.issn.0366-6964.2018.01.009
24	BHUSHANB,KUMARS U,MATAII,et al.Ferritin nanocages: a novel platform for biomedical applications[J].J Biomed Nanotechnol,2014,10(10):2950-2976. doi: 10.1166/jbn.2014.1980
25	LIZ,CUIK,HUANGK,et al.Self assembling rotavirus VP6 nanoparticle vaccines expressed in Escherichia coli elicit systemic and mucosal responses in mice[J].Protein Pept Lett,2019,26(12):904-909. doi: 10.2174/0929866526666190820161328
26	NAKAMURAM,CROSSW R.The lecithinase (alpha toxin) activity of strains of Clostridium perfringens[J].Proc Soc Exp Biol Med,1968,127(3):719-722. doi: 10.3181/00379727-127-32783
27	谢磊. 抑食金球藻(秦皇岛株)细胞毒性及溶血活性研究[D]. 广州: 暨南大学, 2018.
	XIE L. Cytotoxic effect and hemolytic characteristic of Aureococcus anophagefferens (Qinhangdao strain)[D]. Guangzhou: Jinan University, 2018. (in Chinese)
28	PERTMERT M,ROBERTST R,HAYNESJ R.Influenza virus nucleoprotein-specific immunoglobulin G subclass and cytokine responses elicited by DNA vaccination are dependent on the route of vector DNA delivery[J].J Virol,1996,70(9):6119-6125. doi: 10.1128/jvi.70.9.6119-6125.1996
29	JEWELLS A,TITBALLR W,HUYETJ,et al.Clostridium perfringens α-toxin interaction with red cells and model membranes[J].Soft Matter,2015,11(39):7748-7761. doi: 10.1039/C5SM00876J
30	HUJ,CLADELN M,CHRISTENSENN D.Increased immunity to cottontail rabbit papillomavirus infection in EIII/JC inbred rabbits after vaccination with a mutant E6 that correlates with spontaneous regression[J].Viral Immunol,2007,20(2):320-325. doi: 10.1089/vim.2006.0104
31	NAGAHAMAM,NAKAYAMAT,MICHIUEK,et al.Site-specific mutagenesis of Clostridium perfringens alpha-toxin: replacement of Asp-56, Asp-130, or Glu-152 causes loss of enzymatic and hemolytic activities[J].Infect Immun,1997,65(8):3489-3492. doi: 10.1128/iai.65.8.3489-3492.1997
32	NAGAHAMAM,OKAGAWAY,NAKAYAMAT,et al.Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin[J].J Bacteriol,1995,177(5):1179-1185. doi: 10.1128/jb.177.5.1179-1185.1995
33	ZAKERIB,FIERERJ O,CELIKE,et al.Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin[J].Proc Natl Acad Sci U S A,2012,109(12):E690-E697.
34	赵晓军,王美玲,段跃强,等.重组产气荚膜梭菌α毒素对家兔的安全性和免疫效力评价[J].中国预防兽医学报,2023,45(4):429-433.
	ZHAOX J,WANGM L,DUANY Q,et al.Safety andimmune efficacy of recombinant Clostridium perfringens α-toxin in rabbit[J].Chinese Journal of Preventive Veterinary Medicine,2023,45(4):429-433.
35	杜吉革,朱真,薛麒,等.产气荚膜梭菌重组ε毒素突变体的免疫保护力评价[J].畜牧兽医学报,2018,49(4):777-785. doi: 10.11843/j.issn.0366-6964.2018.04.015
	DUJ G,ZHUZ,XUEQ,et al.Evaluation of protective efficacy of recombinant mutant of Clostridium perfringens ε toxin[J].Acta Veterinaria et Zootechnica Sinica,2018,49(4):777-785. doi: 10.11843/j.issn.0366-6964.2018.04.015
36	KANGY F,SUNC,ZHUANGZ,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. doi: 10.1021/acsnano.0c08379
37	MAASSENC B,BOERSMAW J,VAN HOLTEN-NEELENC,et al.Growth phase of orally administered Lactobacillus strains differentially affects IgG1/IgG2a ratio for soluble antigens: implications for vaccine development[J].Vaccine,2003,21(21-22):2751-2757. doi: 10.1016/S0264-410X(03)00220-2
38	TANGX,YUW,SHENL,et al.Conjugation with 8-arm PEG and CRM (197) enhances the immunogenicity of SARS-CoV-2 ORF8 protein[J].Int Immunopharmacol,2022,109,108922. doi: 10.1016/j.intimp.2022.108922
39	YENYUWADEES,SANCHEZ-TRINCADO LOPEZJ L,SHAHR,et al.The evolving role of tissue-resident memory T cells in infections and cancer[J].Sci Adv,2022,8(33):eabo5871. doi: 10.1126/sciadv.abo5871

基因名称 Gene name	引物名称 Primer name	引物序列(5′→3′) Primer sequence(5′→3′)
α	α-F	GCAGCAGCCATCATCATCATCATCACTGGGA$\underline{{\rm{TGGAAAGAT}}}$
α	α-R	CTCAGCTTCCTTTCGGGCTTTGTTA$\underline{{\rm{TTATTTTATATTATAAG}}}$
α_m2	α_m2-F	$\underline{{\rm{TAAGAATGCATATGATCTATATC}}}$AAGATGGTTTCTGGGATCCTG
α_m2	α_m2-R	$\underline{{\rm{GATATAGATCATATGCATTCTTATC}}}$ATAACCTGGATAAGTAGAACC
α_m2ST	α-ST-F	GTGGTGGTGGCAGTGCACATATTGTTATGGTTGATGCGTATAAACCGACAAAA $\underline{{\rm{TAATAACAAAGCCCG}}}$
α_m2ST	α-ST-R	CACTACCACCGCCACCGCTACCACCACCACC $\underline{{\rm{TTTTATATTATAAGTTGAATTTCCTGAAATCCACTC}}}$

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Preparation of Clostridium perfringens α Toxin-ferritin Nanoparticle Antigens and Evaluation of Its Immunogenicity in Mice

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