产气荚膜梭菌α毒素C末端的二拷贝串联表达与免疫原性分析
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摘要
本研究旨在获得产气荚膜梭菌α毒素(CPA)C末端(第247-370位氨基酸,CPA_C)二拷贝串联融合蛋白,并评价其免疫原性。对已知的A型产气荚膜梭菌CPA_C编码基因进行优化设计,将其以同向串连的方式串联成二拷贝基因(GCPA_(C2)),经人工合成获得基因片段GCPA_(C2)。将GCPA_(C2)克隆至原核表达载体pET-30a (+)中进行表达与纯化,获得重组蛋白rCPA_(C2)。利用Western Blot方法检测rCPA_(C2)与A型产气荚膜梭菌毒素抗血清的反应性。随后,用rCPA_(C2)免疫家兔,并检测一免及二免后兔血清的中和抗体效价。在二免21 d后,经耳缘静脉注射1个家兔MLD的A型产气荚膜梭菌毒素,检测rCPA_(C2)对家兔的免疫保护效果。结果表明,rCPA_(C2)主要以包涵体的形式表达且能与A型产气荚膜梭菌毒素抗血清反应。每毫升的一免和二免抗血清分别可中和30个和80个小鼠MLD的A型产气荚膜梭菌毒素。1个兔MLD的A型产气荚膜梭菌毒素攻毒后,对照组4/4死亡,免疫组得到了100%(4/4)的保护。以上结果说明,rCPA_(C2)具有良好的免疫原性,从而为A型产气荚膜梭菌病基因工程疫苗的研制提供了重要的实验数据。
This study was conducted to obtain a tandem fusion protein of two copies of CPA C-terminal(CPA_C) and to evaluate the immunogenicity of it. Based on the known CPA sequence of Clostridium perfringens type A, gene of CPA_C was optimized. Two copies of CPA_C genes, GCPA_(C2) were tandemly linked in the same direction and synthesized. Then,GCPA_(C2) was cloned into prokaryotic expression vector pET-30 a(+) for expression and purification to get the recombiant protein, rGCPA_(C2). Reactivity of rGCPA_(C2) with antiserum of Clostridium perfringens type A was detected by Western blot. Rabbits were immunized with rGCPA_(C2) to prepare antiserum and detect the neutralizing titer. The results showed that rGCPA_(C2) was presented predominantly in an insoluble form(inclusion bodies), and it could react with the antiserum of Clostridium perfringens type A. After the first immunization, sera from rabbits immunized with rGCPA_(C2 )could neutralize 30 mice MLD Clostridium perfringens type A toxin per ml, and 80 mice MLD after twice immunization. Moreover, rabbits in rGCPA_(C2)immunized group fully survived at the dose of 1 rabbit MLD of Clostridium perfringens type A toxin challenge, where as all of the rabbits died(4/4) in the control groups. These data suggest that rGCPA_(C2 )is a potential vaccine candidate for genetic engineering subunit vaccine of Clostridium perfringens type A.
This study was conducted to obtain a tandem fusion protein of two copies of CPA C-terminal(CPA_C) and to evaluate the immunogenicity of it. Based on the known CPA sequence of Clostridium perfringens type A, gene of CPA_C was optimized. Two copies of CPA_C genes, GCPA_(C2) were tandemly linked in the same direction and synthesized. Then,GCPA_(C2) was cloned into prokaryotic expression vector pET-30 a(+) for expression and purification to get the recombiant protein, rGCPA_(C2). Reactivity of rGCPA_(C2) with antiserum of Clostridium perfringens type A was detected by Western blot. Rabbits were immunized with rGCPA_(C2) to prepare antiserum and detect the neutralizing titer. The results showed that rGCPA_(C2) was presented predominantly in an insoluble form(inclusion bodies), and it could react with the antiserum of Clostridium perfringens type A. After the first immunization, sera from rabbits immunized with rGCPA_(C2 )could neutralize 30 mice MLD Clostridium perfringens type A toxin per ml, and 80 mice MLD after twice immunization. Moreover, rabbits in rGCPA_(C2)immunized group fully survived at the dose of 1 rabbit MLD of Clostridium perfringens type A toxin challenge, where as all of the rabbits died(4/4) in the control groups. These data suggest that rGCPA_(C2 )is a potential vaccine candidate for genetic engineering subunit vaccine of Clostridium perfringens type A.
引文
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[15] Hoang T H,Hong H A,Clark G C,et al.Recombinant Bacillus subtilis expressing the Clostridium perfringens alpha toxoid is a candidate orally delivered vaccine against necrotic enteritis[J].Infect Immun,2008,76(11):5 257-5 265.
[16] 中国兽药典委员会.中华人民共和国兽药典(2015年版三部)[M].北京:中国农业出版社,2016:45-46.
[17] 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.
[18] Li Q,Xin 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):2 386-2 392.
[19] 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].Veterinary Journal,2016,217:89-94.
[20] Nagahama M,Okagawa Y,Nakayama T,et al.Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin[J].J.Bacteriol,1995,177:1 179-1 185.
[2] 郑晓丽,窦贤明,胡道俊,等.产气荚膜梭菌对养牛业的危害及其防制[J].中国畜牧兽医,2010,37(8):211-214.
[3] Lebrun M,Mainil J G,Linden A.Cattle enterotoxaemia and Clostridium perfringens:description,diagnosis and prophylaxis[J].Veterinary Record,2010,167(1):13-22.
[4] Canard B,Cole S.Genome organization of the anaerobic pathogen Clostridium perfringens[J].Proc Natl Acad Sci USA,1989,86:6 676-6 680.
[5] Sakurai J,Nagahama M,Oda M.Clostridium perfringens alpha-toxin:characterization and mode of action[J].J Biochem,2004,136:569-574.
[6] Oda M,Terao Y,Sakurai J,Nagahama M.Membrane-binding mechanism of Clostridium perfringens alpha-toxin[J].Toxins,2015,7:5 268-5 275.
[7] Goossens E,Verherstraeten S,Valgaeren B R,et al.The C-terminal domain of Clostridium perfringens alpha toxin as a vaccine candidate against bovine necrohemorrhagic enteritis[J].Vet Res,2016,47.
[8] Byrne M P,Smith L A.Development of vaccines for prevention of botulism[J].Biochimie,2000,82:955-966.
[9] Titball R W.Gas gangrene:An open and closed case[J].Microbiology,2005,151:2 821-2 828.
[10] Kulkarni R R,Parreira V R,Sharif S,et al.Immunization of broiler chickens against Clostridium perfringens alpha-induced necrotic enteritis[J].Clin Vaccine Immunol,2007,14:1 070-1 077.
[11] Williamson E D,Titball R W.A genetically engineered vaccine against the alpha-toxin of Clostridium perfringens protects mice against experimental gas gangrene[J].Vaccine,1993,11:1 253-1 258.
[12] Neeson B N,Clark G C,Atkins H S,et al.Analysis of protection afforded by a Clostridium perfringens alpha-toxoid against heterologous clostridial phospholipases C[J].Microb Pathog,2007,43:161-165.
[13] Nagahama M,Oda M,Kobayashi K,et al.A recombinant carboxy-terminal domain of alpha-toxin protects mice against Clostridium perfringens[J].Microbiol Immunol,2013,57:340-345.
[14] Bennett A M,Lescott T,Phillpotts R J,et al.Recombinant vaccinia viruses protect against Clostridium perfringens alpha-toxin[J].Viral Immunol,1999,12(2):97-105.
[15] Hoang T H,Hong H A,Clark G C,et al.Recombinant Bacillus subtilis expressing the Clostridium perfringens alpha toxoid is a candidate orally delivered vaccine against necrotic enteritis[J].Infect Immun,2008,76(11):5 257-5 265.
[16] 中国兽药典委员会.中华人民共和国兽药典(2015年版三部)[M].北京:中国农业出版社,2016:45-46.
[17] 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.
[18] Li Q,Xin 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):2 386-2 392.
[19] 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].Veterinary Journal,2016,217:89-94.
[20] Nagahama M,Okagawa Y,Nakayama T,et al.Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin[J].J.Bacteriol,1995,177:1 179-1 185.