产气荚膜梭菌重组ε毒素突变体的免疫保护力评价

doi:10.11843/j.issn.0366-6964.2018.04.015

Abstract

Abstract:

This experiment was conducted to obtain recombinant mutant of Clostridium perfringens ε toxin and subsequently evaluate the virulence and immunogenicity of the recombinant toxin. The ε toxin gene of C. perfringens type D strain using optimized codons was synthesized based on the sequence reported. At the same time, three amino acid mutations:Y30 and Y196 substituted with alanine, H106 substituted with proline, were introduced into this sequence synthesized. Then,this ε toxin gene was cloned into prokaryotic expression vector pET3a- (+) to construct a recombinant E. coli, followed by induction with IPTG to yield the recombinant protein,following with purification. The reactivity of the purified protein with antiserum of C. perfringens type D was determined by Western blot and the mouse was used to evaluate the virulence of purified protein. The rabbit antiserum against the recombinant proteins was prepared and the neutralizing titer was measured according to the method prescribed in Chinese Veterinary Pharmacopoeia (2015). The results showed that recombinant protein was expressed at a high level in a soluble form with a ratio of about 40% by gray scale scanning, And the protein could react with the antiserum of C. perfringens type D. The protein with the injection volume of 6.25×10⁶ ng·kg^-1 still shows no virulence to mouse. The titer of rabbit antiserum against Clostridium perfringens type D could reach 50-60 and 400-450 Minimum Lethal Dose (MLD) after the first and second immunization and respectively.After challenge with 1MLD of C. perfringens type D toxin, all the rabbits immunized with recombinant protein were protected. The results suggest that the recombinant mutant of C. perfringens ε toxin without virulence retains the good immunogenic antigen, which provides important experimental data for the development of novel C. perfringens vaccine.

CLC Number:

S852.616.3

DU Ji-ge, ZHU Zhen, XUE Qi, LI Qi-hong, YIN Chun-sheng, PENG Xiao-bing, YAO Wen-sheng, KANG Kai, CHEN Xiao-yun. Evaluation of Protective Efficacy of Recombinant Mutant of Clostridium perfringens ε Toxin[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(4): 777-785.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2018.04.015

https://www.xmsyxb.com/EN/Y2018/V49/I4/777

References

[1] MCCLANE B A, UZAL F A, MIYAKAWA M E, et al. The enterotoxic clostridia[M]//FALKOW S, DWORKIN M, ROSENBURG E, et al. The Prokaryotes. New York, NY, USA:Springer, 2006:698-752.
[2] HATHEWAY C L. Toxigenic clostridia[J]. Clin Microbiol Rev,1990, 3(1):66-98.
[3] SONGER J G. Clostridial enteric diseases of domestic animals[J]. Clin Microbiol Rev, 1996, 9(2):216-234.
[4] NOVAK J S, JUNEJA V K. Clostridium perfringens:hazards in new generation foods[J]. Innov Food Sci Emerg Technol,2002, 3(2):127-132.
[5] UZAL F A, FREEDMAN J C, SHRESTHA A, 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.
[6] DECKER M, DE ALMEIDA GOMES G, GALVO A C, et al. Evaluation of a new mathematical model to describe Clostridium perfringens growth during the cooling of cooked ground beef[J]. Food Sci Technol, 2013, 33(3):507-512.
[7] FERREIRA M R A, MOREIRA G M S G, DA CUNHA C E P, et al. Recombinant Alpha, Beta, and epsilon toxins of Clostridium perfringens:production strategies and applications as veterinary vaccines[J]. Toxins, 2016, 8(11):340-363.
[8] 王莹莹,乔艺然,赵蕾, 等. 产气荚膜梭菌重组NetB毒素细胞毒性作用研究[J]. 畜牧兽医学报, 2016, 47(5):1018-1025.
WANG Y Y, QIAO Y R, ZHAO L, et al. The study on cytotoxicity of recombinant NetB toxin of Clostridium perfringens[J]. Acta Veterinatria et Zootechnica Sinica, 2016, 47(5):1018-1025. (in Chinese)
[9] 柳美玲, 王爱华, 陈凤梅, 等. 浅析牛产气荚膜梭菌病[J]. 山东畜牧兽医, 2015(2):59-61.
LIU M L, WANG A H, CHEN F M, et al. Clostridium perfringens in cattle industry[J]. Shandong Journal of Animal Science and Veterinary Medicine, 2015(2):59-61. (in Chinese)
[10] 钏有科, 肖啸, 濮永华, 等. 努比亚山羊产气荚膜梭菌病的诊治[J]. 中国兽医杂志, 2014, 50(12):42-43.
CHUAN Y K, XIAO X, PU Y H, et al. Diagnosis and treatment of Clostridium perfringens infection in Nubian goats[J]. Chinese Journal of Veterinary Medicine, 2014, 50(12):42-43. (in Chinese)
[11] VAN IMMERSEEL F, DE BUCK J, PASMANS F, et al. Clostridium perfringens in poultry:an emerging threat for animal and public health[J]. Avian Pathol, 2004, 33(6):537-549.
[12] 郑晓丽, 宋振银, 倪学勤. 产气荚膜梭菌对家禽业的危害及其预防[J]. 中国家禽, 2008, 30(24):69-71.
ZHENG X L, SONG Z Y, NI X Q. Emerging threat and prevention of Clostridium perfringens in poultry industry[J]. China Poultry, 2008, 30(24):69-71. (in Chinese)
[13] REVITT-MILLS S A, ROOD J I, ADAMS V. Clostridium perfringens extracellular toxins and enzymes:20 and counting[J]. Microbiol Aust, 2015, 36(3):114-117.
[14] NⅡLO L. Clostridium perfringens in animal disease:A review of current knowledge[J]. Can Vet J, 1980, 21(5):141-148.
[15] LEBRUN M, MAINIL J G, LINDEN A. Cattle enterotoxaemia and Clostridium perfringens:description, diagnosis and prophylaxis[J]. Vet Rec,2010, 167(1):13-22.
[16] SAYEED S, LI J H, MCCLANE B A. Virulence plasmid diversity in Clostridium perfringens type D isolates[J]. Infect Immun, 2007, 75(5):2391-2398.
[17] SAKURAI J. Toxins of Clostridium perfringens[J]. Rev Med Microbiol, 1995, 6(3):175-185.
[18] HUNTER S E, CLARKE I N, KELLY D C, et al. Cloning and nucleotide sequencing of the Clostridium perfringens epsilon-toxin gene and its expression in Escherichia coli[J]. Infect Immun, 1992, 60(1):102-110.
[19] MINAMI J, KATAYAMA S, MATSUSHITA O, et al. Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N-and C-terminal peptides[J]. Microbiol Immunol, 1997, 41(7):527-535.
[20] FREEDMAN J C, MCCLANE B A, UZAL F A. New insights into Clostridium perfringens epsilon toxin activation and action on the brain during enterotoxemia[J]. Anaerobe, 2016, 41:27-31.
[21] ALVES G G, DE ÁVILA R A M, CHÁVEZ-OLÓRTEGUI C D, et al. Clostridium perfringens epsilon toxin:the third most potent bacterial toxin known[J]. Anaerobe, 2014, 30:102-107.
[22] COLE A R, GIBERT M, POPOFF M, et al. Clostridium perfringens ε-toxin shows structural similarity to the pore-forming toxin aerolysin[J]. Nat Struct Mol Biol, 2004, 11(8):797-798.
[23] NAGAHAMA M, ODA M, KOBAYASHI K, et al. A recombinant carboxy-terminal domain of alpha-toxin protects mice against Clostridium perfringens[J]. Microbiol Immunol, 2013, 57(5):340-345.
[24] DAS S, MAJUMDER S, KINGSTON J J, et al. Generation and characterization of recombinant bivalent fusion protein r-Cpib for immunotherapy against Clostridium perfringens beta and iota toxemia[J]. Mol Immunol, 2016, 70:140-148.
[25] ALIMOLAEI M, GOLCHIN M, DANESHVAR H. Oral immunization of mice against Clostridium perfringens epsilon toxin with a Lactobacillus casei vector vaccine expressing epsilon toxoid[J]. Infect Genet Evol, 2016, 40:282-287.
[26] LI Q, XIN W W, GAO S, et al. A low-toxic site-directed mutant of Clostridium perfringens ε-toxin as a potential candidate vaccine against enterotoxemia[J]. Hum Vaccin Immunother, 2013, 9(11):2386-2392.
[27] DORCA-ARÉVALO J, PAUILLAC S, DÍAZ-HIDALGO L, et al. Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens[J]. PLoS One, 2014, 9(7):e102417.
[28] YAO W W, KANG J J, KANG L, et al. Immunization with a novel Clostridium perfringens epsilon toxin mutant rETX^Y196E-C confers strong protection in mice[J]. Sci Rep, 2016, 6:24162.
[29] IVIE S E, MCCLAIN M S. Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1[J]. Biochemistry, 2012, 51(38):7588-7595.
[30] BOKORI-BROWN M, SAVVA C G, DA COSTA S P F, et al. Molecular basis of toxicity of Clostridium perfringens epsilon toxin[J]. FEBS J, 2011, 278(23):4589-4601.
[31] 中国兽药典委员会. 中华人民共和国兽药典,2015年版三部[S]. 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 2015 edition[S]. Beijing:China Agriculture Press, 2016:45-46. (in Chinese)
[32] VERHERSTRAETEN S, GOOSSENS E, VALGAEREN B, et al. Non-toxic perfringolysin O and α-toxin derivatives as potential vaccine candidates against bovine necrohaemorrhagic enteritis[J]. Vet J, 2016, 217:89-94.
[33] 李箐. 产气荚膜梭菌α、ε毒素突变体构建及应用[D]. 合肥:安徽医科大学, 2013.
LI Q. Construction of mutants of Clostridium perfringens alpha, epsilon toxin and its application[D]. Hefei:Anhui Medical University, 2013. (in Chinese)
[34] OYSTON P C, PAYNE D W, HAVARD H L, et al. Production of a non-toxic site-directed mutant of Clostridium perfringens ε-toxin which induces protective immunity in mice[J]. Microbiology, 1998, 144(2):333-341.
[35] 农业部兽医局.农业部关于2015年第一期兽药质量监督抽检情况的通报[EB/OL]. (2015-04-02)[2017-12-22]. http://www.moa.gov.cn/zwllm/tzgg/tz/201504/t20150402_4472055.htm. Veterinary Bureau of the Ministry of Agriculture of PRC. Report of the Ministry of agriculture on the inspection of the first phase of the quality supervision of veterinary drugs in 2015[EB/OL]. (2015-04-02)[2017-12-22]. http://www.moa.gov.cn/zwllm/tzgg/tz/201504/t20150402_4472055.htm. (in Chinese)
[36] 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)

Evaluation of Protective Efficacy of Recombinant Mutant of Clostridium perfringens ε Toxin

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 6

Recommended Articles

Metrics

Comments

[1]	ZENG Chengrong, WANG Na, BI Wenwen, MEI Shihui, HE Guangxia, ZHANG Junjie, CHEN Ze, WEN Ming, ZHOU Bijun. Metabonomics Analysis of Duck Ileum Infected by Clostridium perfringens Type A [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2555-2569.
[2]	WU Ke, FENG Hang, WANG Juan, YANG Zengqi. Whole Genome Sequencing and Molecular Characterization Analysis of Clostridium perfringens Type D Strains from Guanzhong Dairy Goats [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3967-3974.
[3]	HUANG Xiaoyu, YANG Qiaoli, YAN Zunqiang, WANG Pengfei, SHI Hairen, GUN Shuangbao. Characterization of circRNA Expression Profiles Involved in Intestines of Clostridium Perfringens Type C-infected Diarrheal Piglet [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 4058-4070.
[4]	ZANG Jianghua, AN Yina, WANG Jing, WANG Kezhi, YANG Jingjing, GAO Min, FENG Landi, TAN Shuyu, HU Yanxin, DONG Yanjun. Investigation on Pathogenicity and Drug Efficacy of Clostridium perfringens Type A [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3561-3569.
[5]	ZHANG Siyu, WANG Yujiong, ZENG Jin. Transcriptome Analysis of Intestinal Injury Induced by Clostridium perfringens Type C Exotoxin in Mouse [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3570-3581.
[6]	WANG Ying-ying，QIAO Yi-ran，ZHAO Lei，LIU Peng，REN Yu-dong，LI Guang-xing，HUANG Xiao-dan，ZHANG Rui-li，YANG Gui-jun. The Study on Cytotoxicity of Recombinant NetB Toxin of Clostridium perfringens [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(5): 1018-1025.