腐败梭菌无毒重组α毒素的表达与免疫保护性分析

doi:10.11843/j.issn.0366-6964.2021.021

Abstract

Abstract: This experiment was conducted to express recombinant Clostridium septicum α toxin without toxin and subsequently evaluate the immunogenicity of the recombinant toxin. Recombinant α toxin gene (GCSA_Δ11) of Clostridium septicum deleted 11 amino acids (212-222) was synthesized based on the sequence reported. Subsequently, GCSA_Δ11 was cloned into the prokaryotic expression vector pET-30a (+) to get the recombinant protein (rCSA_Δ11). The reactivity of rCSA_Δ11 with antiserum of Clostridium septicum crude toxin was detected by Western blot, and mice were used to detect the virulence of rCSA_Δ11. The rabbit antiserum against the recombinant protein was prepared and the neutralizing titer was detected according to the method prescribed in Chinese Veterinary Pharmacopoeia (2015). The results showed that recombinant protein rCSA_Δ11 was expressed at a high level as soluble form with a ratio about 46%, and the protein without virulence to mouse could react with the antiserum of Clostridium septicum toxin; rabbit antiserum after first immunization could neutralize 8-12 mice MLD of Clostridium septicum crude toxin per 0.1 mL, and 32-45 MLD after twice immunizations. After challenge with 1 MLD of Clostridium septicum crude toxin, the rabbits immunized with rCSA_Δ11 were protected with a ration of 100%, whereas all of the rabbits died (4/4) in the control groups. The results suggest that the recombinant of Clostridium septicum α toxin without virulence retains the good immunogenic antigen, which provides important experimental data for the development of Clostridium septicum genetic engineering subunit vaccine.

Key words: Clostridium septicum α toxin, gene deletion, transmembrane domain, no-toxin, immunogenicity

CLC Number:

S855.1

DU Jige, ZHAO Wei, PENG Guorui, LI Qianlin, YIN Chunsheng, YAO Wensheng, ZHANG Xiukun, YANG Liu, FU Lizhi, CHEN Xiaoyun, LIU Ying. Expression and Immunogenicity of No-toxin Recombinant Clostridium septicum α Toxin[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(1): 202-209.

References 30

[1]	GRAY K M, PADILLA P L, SPARKS B, et al. Distant myonecrosis by atraumatic Clostridium septicum infection in a patient with metastatic breast cancer[J]. IDCases, 2020, 20:e00784.
[2]	GAZIOGLU A, KARAGVLLE B, YVKSEL H, et al. Sudden death due to gas gangrene caused by Clostridium septicum in goats[J]. BMC Vet Res, 2018, 14(1):406.
[3]	SASAKI Y, YAMAMOTO K, TAMURA Y, et al. Tetracycline-resistance genes of Clostridium perfringens, Clostridium septicum and Clostridium sordellii isolated from cattle affected with malignant edema[J]. Vet Microbiol, 2001, 83(1):61-69.
[4]	GARCIA J P, MOORE J, LOUKOPOULOS P, et al. Necrotizing gastritis associated with Clostridium septicum in a rabbit[J]. J Vet Diagn Invest, 2014, 26(5):669-673.
[5]	彭国瑞, 彭小兵, 李旭妮, 等. 截短腐败梭菌α毒素的表达及其免疫原性评价[J]. 中国兽医科学, 2019, 49(2):220-225.PENG G R, PENG X B, LI X N, et al. Expression and immunogenicity evaluation of two truncated alpha toxins of Clostridium septicum[J]. Chinese Veterinary Science, 2019, 49(2):220-225. (in Chinese)
[6]	刘旭平, 范永鑫, 母连志, 等. 水貂腐败梭菌感染的诊疗试验[J]. 黑龙江畜牧兽医, 2014(18):122-123, 146.LIU X P, FAN Y X, MU L Z, et al. Trail of diagnosis and treatment of the infection with Clostridium septicum in mink[J]. Heilongjiang Animal Science and Veterinary Medicine, 2014(18):122-123, 146. (in Chinese)
[7]	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 perfringens β toxin and preparation of its antiserum[J]. Animal Husbandry & Veterinary Medicine, 2015, 47(10):93-96. (in Chinese)
[8]	HANG'OMBE M B, MUKAMOTO M, KOHDA T, et al. Cytotoxicity of Clostridium septicum alpha-toxin:its oligomerization in detergent resistant membranes of mammalian cells[J]. Microb Pathog, 2004, 37(6):279-286.
[9]	ROTH F, JANSEN K, PETZKE S. Detection of neutralizing antibodies against α-toxin of different Clostridium septicum strains in cell culture[J]. FEMS Immunol Med Microbiol, 1999, 24(3):353-359.
[10]	AMIMOTO K, OHGITANI T, SASAKI O, et al. Protective effect of Clostridium septicum alpha-toxoid vaccine against challenge with spores in guinea pigs[J]. J Vet Med Sci, 2002, 64(1):67-69.
[11]	BALLARD J, BRYANT A, STEVENS D, et al. Purification and characterization of the lethal toxin (Alpha-Toxin) of Clostridium septicum[J]. Infect Immun, 1992, 60(3):784-790.
[12]	BALLARD J, CRABTREE J, ROE B A, et al. The primary structure of Clostridium septicum alpha-toxin exhibits similarity with that of Aeromonas hydrophila aerolysin[J]. Infect Immun, 1995, 63(1):340-344.
[13]	POPOFF M R, BOUVET P. Clostridial toxins[J]. Future Microbiol, 2009, 4(8):1021-1064.
[14]	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.
[15]	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.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. (in Chinese)
[16]	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 G, XUE Q, ZHU Z, et al. Evaluation of protective efficacy of recombinant mutant of Clostridium septicum α toxin[J]. Chinese Veterinary Science, 2018, 48(9):1124-1130. (in Chinese)
[17]	TWETEN R K. Clostridium perfringens beta toxin and Clostridium septicum alpha toxin:their mechanisms and possible role in pathogenesis[J]. Vet Microbiol, 2001, 82(1):1-9.
[18]	KNAPP O, MAIER E, MKADDEM S B, et al. Clostridium septicum alpha-toxin forms pores and induces rapid cell necrosis[J]. Toxicon, 2010, 55(1):61-72.
[19]	GORDON V M, BENZ R, FUJII K, et al. Clostridium septicum alpha-toxin is proteolytically activated by furin[J]. Infect Immun, 1997, 65(10):4130-4134.
[20]	HONG Y, OHISHI K, INOUE N, et al. Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum α-toxin[J]. EMBO J, 2002, 21(19):5047-5056.
[21]	KENNEDY C L, SMITH D J, LYRAS D, et al. Programmed cellular necrosis mediated by the pore-forming α-toxin from Clostridium septicum[J]. PLoS Pathog, 2009, 5(7):e1000516.
[22]	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.
[23]	MELTON-WITT J A, BENTSEN L M, TWETEN R K. Identification of functional domains of Clostridium septicum alpha toxin[J]. Biochemistry, 2006, 45(48):14347-14354.
[24]	中国兽药典委员会. 中华人民共和国兽药典-三部[M].2015年版北京:中国农业出版社, 2016:45-46.Commission of Chinese Veterinary Pharmacopoeia. Veterinary pharmacopoeia of the people's repubilc of China volume Ⅲ[M].2015 ed. Beijing:China Agricultural Press, 2016:45-46. (in Chinese).
[25]	AMIMOTO K, SASAKI Y, FUKUYAMA S I, et al. Genetic variation and cross-reactivity of Clostridium septicum alpha-toxin[J]. Vet Microbiol, 2006, 114(1-2):51-59.
[26]	彭国瑞, 蒋玉文, 彭小兵, 等. 腐败梭菌α毒素的研究进展[J]. 中国兽药杂志, 2013, 47(10):53-56.PENG G R, JIANG Y W, PENG X B, et al. Research progress of Clostridium septicum Alpha-toxin[J]. Chinese Journal of Veterinary Drug, 2013, 47(10):53-56. (in Chinese)
[27]	张燕, 边艳青, 赵宝华. 腐败梭菌α毒素基因的克隆表达及其类毒素的免疫原性研究[J]. 生物工程学报, 2007, 23(1):67-72.ZHANG Y, BIAN Y Q, ZHAO B H. The study on the cloning and expression of alpha toxin gene of Clostridium septicum toxoid and the immunity of the toxoid[J]. Chinese Journal of Biotechnology, 2007, 23(1):67-72. (in Chinese)
[28]	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 α-toxin[D]. Beijing:China Institute of Veterinary Drug Control, 2014. (in Chinese)
[29]	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 Y. Biological characteristics and immunogenicity of recombinant Clostridium septicum alpha toxin mutants[D]. Beijing:China Institute of Veterinary Drug Control, 2019. (in Chinese)
[30]	SHIN D J, LEE J J, CHOY H E, et al. Generation and characterization of Clostridium septicum α toxin mutants and their use in diagnosing paroxysmal nocturnal hemoglobinuria[J]. Biochem Biophys Res Commun, 2004, 324(2):753-760.

Expression and Immunogenicity of No-toxin Recombinant Clostridium septicum α Toxin

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 30

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	CHENG Tianyin, WU Congying, LIU Yuke, DUAN Deyong. Studies on the Role of HSP70-b2 and Its 14-Mer-like Peptide from Haemaphysalis flava [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2570-2580.
[2]	HE Shufan, ZOU Yuantong, HUANG Zhilan, LI Qian, JIANGCUO Wengxi, YUE Hua, TANG Cheng, LIU Jie. Detection of Bovine Adenovirus Type 3 Infection Status and Its Fiber Shaft Gene [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1333-1340.
[3]	ZHANG Jiaqi, JIA Xining, ZHOU Qun, SONG Xin, ZHU Chenxi, LI Gege, ZHANG Bin. Construction of BVDV-1 Virus-like Particles based on E2 Protein and Evaluation of Its Immune Efficacy in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5143-5153.
[4]	LI Chang, ZHOU Ping, WEN Ping, YANG Keli, LIU Wei, GUO Rui, LIU Zewen, YUAN Fangyan, GAO Ting, SONG Haofei, TIAN Yongxiang, ZHOU Danna. Investigation of the Genome-wide Sequence and Immunogenicity of Capsid Protein in Mice for a Porcine Circovirus 2d Genotype Strain [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4289-4299.
[5]	YANG Fuchun, LIU Rui, LI Xiaohan, GAO Li, LIU Changjun, QI Xiaole, CUI Hongyu, WANG Xiaomei, GAO Yulong, LI Kai. Construction of UL41 Gene Deletion Strain of Duck Enteritis Virus and Analysis of Its Replication Ability in vitro [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2689-2696.
[6]	YUE Yahui, XING Xiaoyong, WU Xiaochun, WEN Fengqin, ZHANG Hongyan, LONG Cuiqin, ZHANG Li, MA Haiyun, BAO Shijun. Expression of Elongation Factor Thermo Unstable of Mycoplasma synoviae WVU1853 Strain and Analysis of Its Adherence Characterization [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2): 520-528.
[7]	LI Yajun, RU Yi, HAO Rongzeng, JIANG Chenghui, WANG Wei, ZHANG Yue, ZHANG Guicai, LIU Huanan, LU Bingzhou, YANG Yang, TAO Shiyu, YANG Rui, SONG Xiangdong, CHEN Jiao, YU Sijiu, ZHENG Haixue. Expression of Bovine Viral Diarrhea Virus E2 Protein in CHO Cells and Immunogenicity Analysis [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4315-4324.
[8]	LIU Junwen, WU Qiongjuan, XING Gang, ZHAN Songhe, WEI Jianzhong, SUN Pei, LIU Xuelan, LI Yu. Immunogenicity Analysis and Protective Effects of CbpB Protein of Erysipelothrix rhusiopathiae in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(6): 1689-1699.
[9]	CHEN Shengli, HAO Huafang, JI Wenheng, CHU Yuefeng. Research Advances in Immunogenicity of Related Proteins of Animal Mycoplasmas [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1230-1237.
[10]	LÜ Fenfen, MA Xiaohui, WANG Li, MA Chen, ZHANG Baojiang, SU Yan. Immunogenicity Analysis of Three Different Antigen Proteins of Streptococcus equi in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(3): 752-762.
[11]	SHI Lei, ZHOU Yahong, TIAN Zhancheng, GAO Shandian, DU Junzheng, GUAN Guiquan, LIU Guangyuan, LUO Jianxun, YIN Hong. Generation of Genotype Ⅰ Japanese Encephalitis Virus Virus-like Particles and Evaluation of Its Immunogenicity in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1391-1398.
[12]	WU Leyi, WU Sufang, CHE Ying, ZHANG Qiang, LI Peng, BIAN Guanglin, WANG Xuannian. The Establishment of CHO-K1 Cell Line Stably Expressing High Level PCV2-Cap and Immunogenicity Analysis [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(5): 1056-1063.
[13]	FENG Yao, YUE Hui-xi, LIU Hong-ming, CAO San-jie, WU Rui, HUANG Xiao-bo, WEN Yi-ping, ZHAO Qin, WEN Xin-tian. The Immunogenicity Evaluation of Inactivated Vaccines of Swine Japanese Encephalitis Virus Genetype Ⅰ [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(5): 889-895.
[14]	YAO Yan-bin，LU Ping， YANG Zhi-peng， WEI Jian-zhong，SUN Pei，LI Yu. Prokaryotic Expression and Immunogenicity Analysis of SpaA Gene in Erysipelothrix rhusiopathiae [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(3): 492-500.
[15]	WU Chang-hua，CHEN Shao-pin，WEN Gui-lan，LI Chen，LI Tian-zhen，LIN Han-qing，GUAN Guo-dan，WANG Kai-gong，WEN Ming，GONG Xin-yong. Prokaryotic Expression of the LSm14A Gene in the Frizzle Chicken Based on the Sequence and Immunogenicity Analyses of the Expressed LSm14A [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(7): 1511-1516.