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

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (9): 4626-4637.doi: 10.11843/j.issn.0366-6964.2025.09.040

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

赵慧玉¹^,²(), 雷伊诺²^,³(), 幸倩如², 张珊², 张广智², 蒋卉², 沈青春², 丁家波², 姜世金¹^,*(), 范学政²^,*()

1. 山东农业大学, 泰安 271000
2. 中国农业科学院北京畜牧兽医研究所, 北京 100193
3. 广西大学, 南宁 530004

收稿日期:2024-12-18 出版日期:2025-09-23 发布日期:2025-09-30
通讯作者: 姜世金,范学政 E-mail:1765137226@qq.com;1229042392@qq.com;jshijin@163.com;fanxuezheng@caas.cn
作者简介:赵慧玉(1999-)，女，山东莒南人，硕士，主要从事动物微生物学和免疫学研究，E-mail：1765137226@qq.com
雷伊诺(2000-)，女，山西长治人，硕士，主要从事动物微生物学和免疫学研究，E-mail：1229042392@qq.com
第一联系人：
赵慧玉和雷伊诺为同等贡献作者
基金资助:
宁夏重点研发项目(2024BBF02014)；中国农科院创新工程重点项目(CAAS-CSLPDCP-202403)

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

ZHAO Huiyu¹^,²(), LEI Yinuo²^,³(), XING Qianru², ZHANG Shan², ZHANG Guangzhi², JIANG Hui², SHEN Qingchun², DING Jiabo², JIANG Shijin¹^,*(), FAN Xuezheng²^,*()

1. Shandong Agricultural University, Tai'an 271000, China
2. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
3. Guangxi University, Nanning 530004, China

Received:2024-12-18 Online:2025-09-23 Published:2025-09-30
Contact: JIANG Shijin, FAN Xuezheng E-mail:1765137226@qq.com;1229042392@qq.com;jshijin@163.com;fanxuezheng@caas.cn

摘要/Abstract

摘要：

本研究旨在制备产气荚膜梭菌α毒素-铁蛋白纳米颗粒抗原并评价其对小鼠的免疫原性。将带有D56G、H68G点突变和Spytag序列的α毒素全长基因和带有SpyCatcher序列的铁蛋白(Ferritin)基因片段分别克隆至原核表达载体pET 28a，后进行表达并对表达产物进行鉴定和纯化。对纯化后的α_m2ST进行细胞毒性、卵磷脂酶活性以及溶血活性分析。将纯化的α_m2ST和FeSC在体外对接，随后，将α毒素-铁蛋白(α_m2-Fe)对接产物和α_m2ST分别与佐剂混合后免疫小鼠。免疫后每周采集小鼠血清，检测IgG、IgG亚型抗体水平和中和效价，并在二免后28 d取脾细胞分析T细胞亚群和IFN-γ分泌水平。结果显示，在28 ℃条件下，α_m2ST和FeSC均以可溶性表达为主。α_m2ST未被检测出细胞毒性、卵磷脂酶活性和溶血活性，这说明α_m2ST已无毒性。将α_m2ST和FeSC体外对接后，经SDS-PAGE、TEM和DLS分析，证实α_m2-Fe形成直径32.7~50.7 nm的纳米颗粒，主峰为37.8 nm。小鼠免疫后，α_m2-Fe组能产生较高水平的IgG2a抗体(P < 0.001)；小鼠二免后21 d，α_m2-Fe组血清中和效价为64倍，显著高于α_m2ST组的32倍(P < 0.05)；二免后28 d，α_m2-Fe组脾细胞刺激后上清中的IFN-γ也高于α_m2ST组(P < 0.05)，流式细胞术分析显示α_m2-Fe组产生了较高比例的效应性CD8⁺ T细胞(P < 0.000 1)。小鼠试验证实α_m2-Fe纳米颗粒抗原能有效刺激小鼠产生体液免疫和细胞免疫。

关键词: 产气荚膜梭菌, α毒素, 铁蛋白, 纳米颗粒, 免疫原性

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

中图分类号:

S852.616.3

赵慧玉, 雷伊诺, 幸倩如, 张珊, 张广智, 蒋卉, 沈青春, 丁家波, 姜世金, 范学政. 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[J]. 畜牧兽医学报, 2025, 56(9): 4626-4637.

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.

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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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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

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

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