产气荚膜梭菌β毒素蛋白抗原表位预测及CPB-N蛋白免疫原性分析
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摘要
为获得具有与β毒素蛋白相同免疫原性的新蛋白,本研究对产气荚膜梭菌β毒素蛋白的氨基酸序列进行综合分析,预测其抗原表位并筛选出有效的新蛋白。采用生物信息学方法综合分析β毒素蛋白二级结构、亲水性、可塑性、抗原性、跨膜区域以及信号肽,同时参考ElliPro服务器在线预测。最终预测aa108～aa320区段为优势抗原表位,在此基础上建立β毒素蛋白三维结构模型,标记特殊位点并筛选出B型产气荚膜梭菌新蛋白(CPB-N)。诱导表达CPB-N后通过SDS-PAGE和western blot鉴定,结果显示在约24 ku处有明显条带。采用间接ELISA方法对免疫小鼠的血清和肠道灌洗液样品进行检测,结果表明CPB-N与β毒素蛋白的免疫原性基本相同。CPB-N蛋白的筛选及预防产气荚膜梭菌引起的疾病奠定了重要的研究基础。
In order to identify a new protein with the same immunogenicity as the beta toxin protein, we comprehensively analyzed the amino acid sequence of Clostridium perfringens the beta toxin protein, predicted the epitope and screened for the new protein with the similar epitope. The bioinformatics method was used to comprehensively analyze the secondary structure,hydrophilicity, surface possibility, plasticity, antigenicity, transmembrane region and signal peptide of the beta toxin protein, and refer to online prediction results for ElliPro servers. Finally, we predicted that the amino acid sequence of the aa108-aa320 was the dominant epitope, established a three-dimensional structural model of the beta toxin protein, labeled a specific site and screened the CPB-N protein. After induction of expression of CPB-N, obvious bands about 24 ku was observed in the identification of 10%SDS-PAGE and western blot. The serum and intestinal lavage fluid samples from immunized mice were tested by indirect ELISA.The results showed that the immunogenicity of CPB-N were essentially same as the beta toxin protein. The selection and confirmation of CPB-N protein provided an important research basis for the prevention of diseases caused by C.perfringens.
In order to identify a new protein with the same immunogenicity as the beta toxin protein, we comprehensively analyzed the amino acid sequence of Clostridium perfringens the beta toxin protein, predicted the epitope and screened for the new protein with the similar epitope. The bioinformatics method was used to comprehensively analyze the secondary structure,hydrophilicity, surface possibility, plasticity, antigenicity, transmembrane region and signal peptide of the beta toxin protein, and refer to online prediction results for ElliPro servers. Finally, we predicted that the amino acid sequence of the aa108-aa320 was the dominant epitope, established a three-dimensional structural model of the beta toxin protein, labeled a specific site and screened the CPB-N protein. After induction of expression of CPB-N, obvious bands about 24 ku was observed in the identification of 10%SDS-PAGE and western blot. The serum and intestinal lavage fluid samples from immunized mice were tested by indirect ELISA.The results showed that the immunogenicity of CPB-N were essentially same as the beta toxin protein. The selection and confirmation of CPB-N protein provided an important research basis for the prevention of diseases caused by C.perfringens.
引文
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[11]Zhao Li,Guo Zhi-hou,Liu Jia-li,et al.Recombinant Lactobacillus casei expressing Clostridium perfringens toxoidsα,β2,εandβ1 gives protection against Clostridium perfringens in rabbits[J].Vaccine,2017,35(32):4010-4021.
[12]李凡飞,张云,李强,等.C型产气荚膜梭菌致病机理研究进展[J].动物医学进展,2016,37(12):82-85.
[13]蔺旭光,李佳怡,娜仁图雅,等.一例小尾寒羊魏氏梭菌的分离鉴定[J].畜牧兽医科技信息,2017,(4):27-28.
[14]霍萍萍,张聪敏,胡明丽,等.产气荚膜梭菌β毒素研究进展[J].动物医学进展,2013,34(9):90-94..
[15]Nagahama M,Hayashi S,Morimitsu S,et al.Biological activities and pore formation of Clostridium perfringens beta toxin in HL 60 cells[J].J Biol Chem,2003,278(38):36934-36941.
[2]Lucey B P,Hutchins G M.William H.Welch,M D,and the discovery of Bacillus welchii[J].Arch Pathol Lab Med,2004,128(10):1193-1195.
[3]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.
[4]孙雨,王晓英,董浩,等.产气荚膜梭菌外毒素基因与相关疫苗的研究进展[J].中国草食动物科学,2016,36(2):58-62.
[5]Anderson R,Huang Yan,Langley J M.Prospects for defined epitope vaccines for respiratory syncytial virus[J].Future Microbiol,2010,5(4):585-602.
[6]宫旭颖,葛俊伟,乔薪瑗,等.产气荚膜梭菌α-β2-ε毒素融合蛋白在大肠杆菌中的表达及免疫原性研究[J].中国预防兽医学报,2015,37(2):128-131.
[7]冶贵生,韩志辉,马玉花,等.C型产气荚膜梭菌β毒素蛋白结构及抗原表位预测[J].甘肃农大学报,2014,53(3):12-16.
[8]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):5257-5265.
[9]曹琛福,梁云浩,花群俊,等.非洲猪瘟病毒p54蛋白抗原表位预测及鉴定[J].中国预防兽医学报,2014,36(11):848-851.
[10]Ponomarenko J,Bui H H,Li Wei,et al.ElliPro:a new structure-based tool for the prediction of antibody epitopes[J].BMCBioinformatics,2008,9(1):514.
[11]Zhao Li,Guo Zhi-hou,Liu Jia-li,et al.Recombinant Lactobacillus casei expressing Clostridium perfringens toxoidsα,β2,εandβ1 gives protection against Clostridium perfringens in rabbits[J].Vaccine,2017,35(32):4010-4021.
[12]李凡飞,张云,李强,等.C型产气荚膜梭菌致病机理研究进展[J].动物医学进展,2016,37(12):82-85.
[13]蔺旭光,李佳怡,娜仁图雅,等.一例小尾寒羊魏氏梭菌的分离鉴定[J].畜牧兽医科技信息,2017,(4):27-28.
[14]霍萍萍,张聪敏,胡明丽,等.产气荚膜梭菌β毒素研究进展[J].动物医学进展,2013,34(9):90-94..
[15]Nagahama M,Hayashi S,Morimitsu S,et al.Biological activities and pore formation of Clostridium perfringens beta toxin in HL 60 cells[J].J Biol Chem,2003,278(38):36934-36941.