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

Abstract

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

CLC Number:

S852.616.3

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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References 30

1	LIGHTY M E , ELVINGER F , EVANS R D , et al. Incidence of clostridial dermatitis (cellulitis) and factors for development of the disease in turkeys[J]. J Appl Poult Res, 2016, 25 (1): 104- 112. doi: 10.3382/japr/pfv065
2	KUHNERT P , CAPAUL S E , NICOLET J , et al. Phylogenetic positions of Clostridium chauvoei and Clostridium septicumbased on 16S rRNA gene sequences[J]. Int J Syst Bacteriol, 1996, 46 (4): 1174- 1176. doi: 10.1099/00207713-46-4-1174
3	KENNEDY C L , LYRAS D , CORDNER L M , et al. Pore-forming activity of alpha-toxin is essential for Clostridium septicum-mediated myonecrosis[J]. Infect Immun, 2009, 77 (3): 943- 951. doi: 10.1128/IAI.01267-08
4	KIRCHNER S , MATZ-RENSING K , DORNER M B , et al. Necrotizing endometritis and isolation of an alpha-toxin producing strain of Clostridium septicum in a wild sooty mangabey from Cote d'Ivoire[J]. J Med Primatol, 2013, 42 (4): 220- 224. doi: 10.1111/jmp.12047
5	雷伊诺. 重组腐败梭菌α毒素原核表达与佐剂对比免疫原性评价[D]. 南宁: 广西大学, 2025.
	LEI Y N. Prokaryotic expression and comparative immunogenicity evaluation of recombinant Clostridium perfringens alpha toxin with different adjuvants[J]. Nanning: Guangxi University, 2025. (in Chinese)
6	AROIAN R , VAN DER GOOT F G . Pore-forming toxins and cellular non-immune defenses (CNIDs)[J]. Curr Opin Microbiol, 2007, 10 (1): 57- 61. doi: 10.1016/j.mib.2006.12.008
7	MOREIRA G M , SALVARANI F M , DA CUNHA C 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
8	彭小兵, 田冬青, 彭国瑞, 等. 产气荚膜梭菌β毒素的表达及其抗血清的制备[J]. 畜牧与兽医, 2015, 47 (10): 93- 96.
	PENG X B , TIAN D Q , PENG G R , et al. Expression of Clostridium septicum β toxin and preparation of its antiserum[J]. Animal Husbandry & Veterinary Medicine, 2015, 47 (10): 93- 96.
9	董令赢. 重组腐败梭菌α毒素突变体的生物学特性及免疫原性研究[D]. 北京: 中国兽医药品监察所, 2019.
	DONG L L. Biological characteristics and immunogenicity of recombinant Clostridium septicum alpha toxin mutants[D]. Beijing: China Institute of Veterinary Drugs Control, 2019. (in Chinese)
10	杜吉革, 薛麒, 朱真, 等. 腐败梭菌α毒素重组突变体的免疫保护力评价[J]. 中国兽医科学, 2018, 48 (9): 1124- 1130.
	DU J , XUE Q , ZHU Z , et al. Evaluation of protective efficacy of recombinant mutant of Clostridium septicumα-toxin[J]. Chinese Veterinary Science, 2018, 48 (9): 1124- 1130.
11	彭国瑞. 重组腐败梭菌α毒素的免疫原性研究[D]. 北京: 中国兽医药品监察所, 2014.
	PENG G R. Immunogenicity of recombinant Clostridium septicum a-toxin[D]. Beijing: China Institute of Veterinary Drugs Control, 2014. (in Chinese)
12	JOHN F A , CRIOLLO V , GAGHAN C , et al. Immunization of turkeys with Clostridium septicum alpha toxin-based recombinant subunit proteins can confer protection against experimental clostridial dermatitis[J]. PLoS One, 2024, 19 (4): e0302555. doi: 10.1371/journal.pone.0302555
13	陈小云, 刘莹, 薛麒, 等. 重组腐败梭菌α毒素对兔的免疫效力分析[J]. 畜牧兽医学报, 2018, 49 (8): 1727- 1734. doi: 10.11843/j.issn.0366-6964.2018.08.018
	CHEN X Y , LIU Y , XUE Q , et al. Evaluation of protective efficacy of recombinant Clostridium septicum α toxin in rabbits[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49 (8): 1727- 1734. doi: 10.11843/j.issn.0366-6964.2018.08.018
14	彭国瑞, 蒋玉文, 彭小兵, 等. 重组腐败梭菌α毒素的生物活性及其灭活疫苗的免疫效果[J]. 中国兽医学报, 2019, 39 (6): 1118- 1124.
	PENG G R , JIANG Y W , PENG X B , et al. Biological activity of recombinant alpha toxin of Clostridium septicum and its inactivated vaccine[J]. Chinese Journal of Veterinary Science, 2019, 39 (06): 1118- 1124.
15	陈小云, 杜吉革, 薛麒, 等. 一种腐败梭菌α毒素重组亚单位疫苗及其生产方法: CN107812183B[P]. 2021-02-02.
	CHEN X Y, DU J G, XUE Q, et al. A recombinant subunit vaccine against Clostridium septicum alpha-toxin and its production method: CN107812183B[P]. 2021-02-02. (in Chinese)
16	杜吉革, 陈小云, 薛麒, 等. 一种腐败梭菌α毒素基因工程疫苗及其生产方法: CN109395072A[P]. 2019-03-01.
	DU J G, CHEN X Y, XUE Q, et al. A genetic engineering vaccine for Clostridium septicum alpha-toxin and its production method: CN109395072A[P]. 2019-03-01. (in Chinese)
17	杜吉革, 陈小云, 薛麒, 等. 一种腐败梭菌α毒素基因工程疫苗及其生产方法: CN109395072B[P]. 2021-11-30.
	DU J G, CHEN X Y, XUE Q, et al. A Clostridium septicum alpha-toxin genetic engineering vaccine and its production method: CN109395072B[P]. 2021-11-30. (in Chinese)
18	杜吉革, 赵炜, 彭国瑞, 等. 腐败梭菌无毒重组α毒素的表达与免疫保护性分析[J]. 畜牧兽医学报, 2021, 52 (1): 202- 209. doi: 10.11843/j.issn.0366-6964.2021.021
	DU J G , ZHAO W , PENG G R , et al. Expression and immunogenicity of no-toxin recombinant Clostridium septicum α toxin[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52 (1): 202- 209. doi: 10.11843/j.issn.0366-6964.2021.021
19	DU J G , MEKI I , LI Q , et al. A non-toxic recombinantClostridium septicum α toxin induces protective immunity in mice and rabbits[J]. Toxicon, 2023, 233, 107234. doi: 10.1016/j.toxicon.2023.107234
20	DING Z , GUAN F , XU G , et al. MPEPE, a predictive approach to improve protein expression in E. coli based on deep learning[J]. Comput Struct Biotechnol J, 2022, 20, 1142- 1153. doi: 10.1016/j.csbj.2022.02.030
21	MELTON J A , PARKER M W , ROSSJOHN J , et al. The identification and structure of the membrane-spanning domain of the Clostridium septicum alpha toxin*[J]. J Biol Chem, 2004, 279 (14): 14315- 14322. doi: 10.1074/jbc.M313758200
22	GORDON V M , NELSON K L , BUCKLEY J T , et al. Clostridium septicum alpha toxin uses glycosylphosphatidylinositol-anchored protein receptors[J]. J Biol Chem, 1999, 274 (38): 27274- 27280. doi: 10.1074/jbc.274.38.27274
23	ALVES M L F , FERREIRA M R A , DONASSOLO R A , et al. Clostridium septicum: A review in the light of alpha-toxin and development of vaccines[J]. Vaccine, 2021, 39 (35): 4949- 4956. doi: 10.1016/j.vaccine.2021.07.019
24	KENNEDY C L , KREJANY E O , YOUNG L F , et al. The α-toxin of Clostridium septicum is essential for virulence[J]. Mol Microbiol, 2005, 57 (5): 1357- 1366. doi: 10.1111/j.1365-2958.2005.04774.x
25	贺笋, 王钢, 张飞, 等. 革兰氏阳性菌表达系统在表达腐败梭菌毒素中的应用、腐败梭菌α毒素的制备方法和疫苗: CN111944838B[P]. 2022-08-16.
	HE S, WANG G, ZHANG F, et al. Application of Gram-positive bacterial expression system in expressing Clostridium septicum toxin, method for preparing Clostridium septicum alpha-toxin, and vaccine: CN111944838B[P]. 2022-08-16. (in Chinese)
26	JABBARI A R , BIDHENDI S M , KHAKI P , et al. Molecular cloning of Clostridium septicum vaccine strain alpha toxin gene in E. coli[J]. Arch Razi Inst, 2014, 69, 15- 20.
27	马清龙, 赵佳慧, 王武斌, 等. 腐败梭菌α毒素蛋白的原核表达及间接ELISA抗体检测方法的建立[J]. 中国兽医科学, 2024, 54 (10): 1309- 1317.
	MA Q L , ZHAO J H , WANG W B , et al. Prokaryotic expression of Clostridium septicum alpha toxin protein and establishment of an indirect ELISA method for detection of anti-α toxin antibody[J]. Chinese Veterinary Science, 2024, 54 (10): 1309- 1317.
28	夏中秋, 赵妍, 王浩, 等. 腐败梭菌α毒素和产气荚膜梭菌ε毒素的融合表达纯化及其免疫效力评价[J]. 黑龙江畜牧兽医, 2022 (15): 115- 119.
	XIA Z Q , ZHAO Y , WANG H , et al. Fusion expression and purification of Clostridium septicum α toxin and Clostridium perfringens ε toxin and its evaluation of immune efficacy[J]. Heilongjiang Animal Science and Veterinary Medicine, 2022 (15): 115- 119.
29	韦良孟, 李昌明, 翁鸿宇, 等. C、D型产气荚膜梭菌和腐败梭菌二价二联类毒素疫苗的创制及其效果评价[J]. 山东畜牧兽医, 2022, 43 (5): 1- 6.
	WEI L M , LI C M , WENG H Y , et al. Research and efficacy evaluation of a bivalent combined toxoid vaccine against Clostridium perfringens types C and D and Clostridium cadaveris[J]. Shandong Journal of Animal Science and Veterinary Medicine, 2022, 43 (5): 1- 6.
30	李昌明. C、D型产气荚膜梭菌类毒素和重组腐败梭菌α毒素二价二联疫苗的制备及其免疫效果评价[D]. 泰安: 山东农业大学, 2020.
	LI C M. Preparation of C, D Clostridium Perfringens toxoid and recombinant Clostridium Septicum alpha toxin bivalent vaccine and evaluation of its immune effect[D]. Tai'an: Shandong Agricultural University, 2020. (in Chinese)

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

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