腐败梭菌α毒素突变体可溶性表达及其对小鼠的免疫评价

doi:10.11843/j.issn.0366-6964.2025.10.034

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (10): 5148-5158.doi: 10.11843/j.issn.0366-6964.2025.10.034

腐败梭菌α毒素突变体可溶性表达及其对小鼠的免疫评价

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

1. 广西大学动物科学技术学院/广西畜禽繁育与疫病防控重点实验室，南宁 530004
2. 中国农业科学院北京畜牧兽医研究所，北京 100193
3. 山东农业大学，泰安 271000

收稿日期:2024-12-17 出版日期:2025-10-23 发布日期:2025-11-01
通讯作者: 韦英益,范学政 E-mail:1229042392@qq.com;1765137226@qq.com;weiyingyi@gxu.edu.cn;fanxuezheng@caas.cn
作者简介:雷伊诺(2000-)，女，山西长治人，硕士，主要从事动物疫病防控技术研究，E-mail：1229042392@qq.com
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
第一联系人：
雷伊诺和赵慧玉为同等贡献作者
基金资助:
宁夏重点研发项目(2024BBF02014)；广西重点研发计划(桂农科AB241484045)；中国农科院创新工程重点项目(CAAS-CSLPDCP-202403)

Soluble Expression of Clostridium septicum Alpha Toxin Mutants and Their Immunological Evaluation in Mice

LEI Yinuo¹^,²(), ZHAO Huiyu²^,³(), XING Qianru², XU Guoshun², ZHANG Guangzhi², ZHANG Shan², JIANG Hui², SHEN Qingchun², DING Jiabo², WEI Yingyi¹^,*(), FAN Xuezheng²^,*()

1. College of Animal Science and Technology, Guangxi University/Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, Nanning, Guangxi 530004
2. Institute of Animal Sciences of Chinese Academy of Agricultural Sciences, Beijing 100193
3. Shandong Agricultural University, Tai′an 271000

Received:2024-12-17 Online:2025-10-23 Published:2025-11-01
Contact: WEI Yingyi, FAN Xuezheng E-mail:1229042392@qq.com;1765137226@qq.com;weiyingyi@gxu.edu.cn;fanxuezheng@caas.cn

摘要/Abstract

摘要：

本研究旨在获得可溶性、免疫原性好的腐败梭菌α毒素突变体。通过MPEPE预测工具对α毒素蛋白进行点突变预测，构建表达载体在大肠杆菌中表达验证，比较rCsa各突变体的可溶性、毒性和小鼠免疫原性。结果表明，rCsa突变体在大肠杆菌中正确表达，条带大小均为约47 ku。rCsaS398D、rCsaK405M、rCsaN397D在大肠杆菌细胞裂解上清中分别占表达总蛋白的9.5%、9.4%、10.5%，在裂解上清与沉淀中的表达量之比分别为2.5、2.6、2.9，rCsaN397D可溶性表达量最高；小鼠毒性试验结果显示3个rCsa突变体毒性均未丢失；Vero细胞毒性试验显示，0.001 6 μg·mL^-1的各rCsa突变体即可引起明显的CPE；ELISA检测rCsa突变体二免21 d后小鼠血清IgG抗体水平，rCsaN397D组血清的S/N值极显著高于rCsaS398D组和rCsaK405M组(P＜0.01)；用Vero细胞测定rCsa突变体二免21 d鼠血清的中和效价，发现rCsaN397D二免21 d鼠血清的中和效价为6400 MLD·mL^-1，极显著高于rCsaS398D和rCsaK405M组免疫血清的中和效价(P＜0.0001)，且高于前期试验中未突变rCsa二免21 d鼠血清的中和效价。综上，rCsaN397D在可溶性、免疫原性方面均优于未突变rCsa、rCsaS398D和rCsaK405M，可作为腐败梭菌病基因工程亚单位疫苗的候选抗原。

关键词: 腐败梭菌α毒素, MPEPE, 毒力, 可溶性, 免疫原性

Abstract:

This study aims to obtain soluble and highly immunogenic mutants of Clostridium septicum alpha toxin. Site mutations in the alpha toxin protein were predicted using the MPEPE prediction tool. Expression vectors were constructed and expressed in E. coli for validation. The solubility, toxicity, and immunogenicity in mice of various rCsa mutants were compared. The results showed that the rCsa mutants were correctly expressed in E. coli, with all bands appearing at approximately 47 ku. The soluble expression levels of rCsaS398D, rCsaK405M, and rCsaN397D accounted for 9.5%, 9.4%, and 10.5% of total expressed proteins in the bacterial lysate supernatant, respectively. The ratios of protein amounts in the supernatant to those in the pellet were 2.5, 2.6, and 2.9, respectively. rCsaN397D showed the highest soluble expression level among the three rCsa mutants, and it was also superior to the 40% solubility of rCsa observed in previous experiments. Mouse toxicity tests indicated that none of the three rCsa mutants lost their toxicity. Vero cell toxicity tests showed that concentrations as low as 0.0016 μg·mL^-1 of each rCsa mutant could induce significant cytopathic effects (CPE). ELISA detection of IgG antibody levels in mouse serum 21 days after the second immunization revealed that the S/N values of the rCsaN397D group serum were significantly higher than those of the rCsaS398D and rCsaK405M groups (P < 0.01). Neutralizing titers of mouse serum from the second immunization tested on Vero cells showed that the neutralizing titer of rCsaN397D was 6400 MLD·mL^-1, which was significantly higher than those of the rCsaS398D and rCsaK405M groups (P < 0.0001), and also higher than the neutralizing titer of unmutated rCsa from previous experiments. In summary, rCsaN397D outperformed the other two rCsa mutants in terms of solubility and immunogenicity, and its immunogenicity was better than that of the unmutated rCsa. Therefore, rCsaN397D can be considered a candidate antigen for a genetically engineered subunit vaccine against Clostridium septicum.

Key words: Clostridium septicumα toxin, MPEPE, virulence, solubility, immunogenicity

中图分类号:

S852.616.3

雷伊诺, 赵慧玉, 幸倩如, 许国顺, 张广智, 张珊, 蒋卉, 沈青春, 丁家波, 韦英益, 范学政. 腐败梭菌α毒素突变体可溶性表达及其对小鼠的免疫评价[J]. 畜牧兽医学报, 2025, 56(10): 5148-5158.

LEI Yinuo, ZHAO Huiyu, XING Qianru, XU Guoshun, ZHANG Guangzhi, ZHANG Shan, JIANG Hui, SHEN Qingchun, DING Jiabo, WEI Yingyi, FAN Xuezheng. Soluble Expression of Clostridium septicum Alpha Toxin Mutants and Their Immunological Evaluation in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 5148-5158.

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突变位点 Mutation sites	ID	AVE (High expression)	STD (High expression)
1	S398D	0.656 729	0.164 954
2	K405M	0.643 722	0.170 202
3	N397D	0.634 557	0.170 353
4	S398N	0.634 438	0.176 428
5	I335P	0.630 136	0.168 943
6	I335E	0.633 71	0.168 512
7	N342A	0.629 691	0.168 727
8	S147D	0.630 924	0.163 245
9	I335T	0.629 425	0.169 731
10	I335Q	0.630 442	0.168 992

引物名称 Primer name	引物序列(5′ → 3′) Primer sequence(5′→3′)
pET28-F	$\underline{{\rm{ATTGATATTAATCACCACCACC}}}$ACCACCACCACTGAGATCCGGCTG
pET28-R	$\underline{{\rm{CCCCCCTCTTCAAGATTTGTAAG}}}$CATGGTATATCTCCTTCTTAAAG
csa-F	$\underline{{\rm{ACTTTAAGAAGGAGATATACCATG}}}$CTTACAAATCTTGAAGAGGGGGG
aN397D-R	$\underline{{\rm{TCAGTGGTGGTGGTGGTGGTG}}}$GTGATTAATATCAATTTTTTTATCATTGTATGTTATGCT-ATCTAGTCTAAATCCTG
aK405M-R	$\underline{{\rm{TCTCAGTGGTGGTGGTGGTG}}}$GTGGTGATTAATATCAATCATTTTATCATTGTATG
aS398D-R	$\underline{{\rm{CAGTGGTGGTGGTGGTGGTG}}}$GTGATTAATATCAATTTTTTTATCATTGTAT-GTTATATCATTTAGTCTAAATCC

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腐败梭菌α毒素突变体可溶性表达及其对小鼠的免疫评价

Soluble Expression of Clostridium septicum Alpha Toxin Mutants and Their Immunological Evaluation in Mice

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