肝片吸虫CatB4蛋白分子特征及其免疫原性分析
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摘要
【目的】明确肝片吸虫组织蛋白酶B4(CatB4)的分子特征及其免疫原性,为研究肝片吸虫CatB4蛋白的生物学特性及揭示其致病机理提供科学依据。【方法】通过RT-PCR扩增肝片吸虫CatB4基因,利用在线分子生物信息学软件对CatB4基因及其编码蛋白进行分子特征分析;构建原核表达载体pET-CatB4,经IPTG诱导获得融合蛋白后进行SDS-PAGE和Western blotting检测分析;并以融合蛋白CatB4免疫小鼠制备多克隆抗体,利用免疫组化对融合蛋白在肝片吸虫体内的表达进行定位分析。【结果】肝片吸虫CatB4基因片段为699 bp,其编码蛋白等电点(pI)7.95,理论分子质量25.89 kD,无信号肽和跨膜区,属于非分泌性蛋白;CatB4蛋白存在10个潜在的磷酸化位点、2个N-糖基化位点和7个N-肉豆蔻酰化位点,有9个抗原决定簇,具有高度保守的CatB结构域,是由无规则卷曲连接9个α-螺旋和8个β-折叠构成其空间结构。基于CatB4基因核苷酸序列同源性构建的系统发育进化树显示,肝片吸虫和大片吸虫聚为一支,二者的同源性为83.98%。SDS-PAGE检测和Western blotting分析结果均显示在41.80 kD处能检测到目的条带。以纯化融合蛋白CatB4与弗氏佐剂乳化后免疫的小鼠均可产生特异性抗体,证实融合蛋白CatB4具有较强的免疫原性;免疫组化定位分析结果显示,在肝片吸虫的卵黄腺腺体细胞、排泄管上皮细胞、肠道上皮细胞和卵黄腺管上皮细胞均发生特异性的抗原抗体反应,呈深棕色。【结论】诱导表达获得的融合蛋白CatB4可特异性识别绵羊肝片吸虫阳性血清,具有较强的免疫原性,且主要在肝片吸虫分泌排泄组织器官中表达,说明CatB4蛋白具有作为肝片吸虫候选抗原的潜力。
【Objective】The aim of the present study was to study the molecular characteristics and immunogenicity of cathepsin B4(CatB4)in Fasciola hepatica,and provide scientific basis for studying the biological characteristics of CatB4 protein and revealing the pathogenic mechanism of F. hepatica.【Method】The F. hepatica CatB4 gene was amplified by RT-PCR and molecular characteristics of its encoded protein was performed by online software. The prokaryotic expression vector p ET-CatB4 was constructed and induced by IPTG to express the fusion protein and then analyzed by SDS-PAGE and Western blotting. Polyclonal antibody was prepared by immunizing mice with fusion protein CatB4 and the protein expression was located in F. hepatica by immunohistochemistry.【Result】The CatB4 gene(699 bp)of F. hepatica was successfully cloned,and its encoded protein isoelectric poin(tpI)was 7.95,theoretical molecular mass was 25.89 kD,no signal peptide and transmembrane region,belonging to non-secretory protein. CatB4 protein had ten potential phosphorylation sites,two N-glycosylation sites and seven N-myristoylation sites. There were nine antigenic determinants with a highly conserved CatB domain,which was a spatial structure composed of random coils connecting nine α-helices and eight β-sheets. A phylogenetic tree based on the nucleotide sequence of the CatB4 gene showed that F. hepatica and F.gigantica were clustered into one branch with homology of 83.98%. SDS-PAGE and Western blotting analysis showed that the target band could be detected at 41.80 k D. The mice after immunization with purified CatB4 protein and Freund's adjuvant could produce specific antibodies,which indicated that the expressed fusion protein CatB4 had good immunogenicity. Immunohistochemical localization analysis showed that specific antigen-antibody reactions occurred in the F. hepatica yolk gland cells,excretory ductal cells,intestinal epithelial cells and yolk glandular epithelial cells,which was dark brown.【Conclusion】The expressed CatB4 fusion protein can specifically recognize the positive serum of F. hepatica,it has strong immunogenicity and can be expressed in the excretory tissues of F. hepatica,which indicates that CatB4 protein has potential as a candidate antigen for F. hepatica.
【Objective】The aim of the present study was to study the molecular characteristics and immunogenicity of cathepsin B4(CatB4)in Fasciola hepatica,and provide scientific basis for studying the biological characteristics of CatB4 protein and revealing the pathogenic mechanism of F. hepatica.【Method】The F. hepatica CatB4 gene was amplified by RT-PCR and molecular characteristics of its encoded protein was performed by online software. The prokaryotic expression vector p ET-CatB4 was constructed and induced by IPTG to express the fusion protein and then analyzed by SDS-PAGE and Western blotting. Polyclonal antibody was prepared by immunizing mice with fusion protein CatB4 and the protein expression was located in F. hepatica by immunohistochemistry.【Result】The CatB4 gene(699 bp)of F. hepatica was successfully cloned,and its encoded protein isoelectric poin(tpI)was 7.95,theoretical molecular mass was 25.89 kD,no signal peptide and transmembrane region,belonging to non-secretory protein. CatB4 protein had ten potential phosphorylation sites,two N-glycosylation sites and seven N-myristoylation sites. There were nine antigenic determinants with a highly conserved CatB domain,which was a spatial structure composed of random coils connecting nine α-helices and eight β-sheets. A phylogenetic tree based on the nucleotide sequence of the CatB4 gene showed that F. hepatica and F.gigantica were clustered into one branch with homology of 83.98%. SDS-PAGE and Western blotting analysis showed that the target band could be detected at 41.80 k D. The mice after immunization with purified CatB4 protein and Freund's adjuvant could produce specific antibodies,which indicated that the expressed fusion protein CatB4 had good immunogenicity. Immunohistochemical localization analysis showed that specific antigen-antibody reactions occurred in the F. hepatica yolk gland cells,excretory ductal cells,intestinal epithelial cells and yolk glandular epithelial cells,which was dark brown.【Conclusion】The expressed CatB4 fusion protein can specifically recognize the positive serum of F. hepatica,it has strong immunogenicity and can be expressed in the excretory tissues of F. hepatica,which indicates that CatB4 protein has potential as a candidate antigen for F. hepatica.
引文
何山红,班克满,郭亚芬,杨丰利,韦英明.2010.广西钦州市郊区水牛大片吸虫感染情况调查分析[J].广西农业科学,41(11):1237-1239.[He S H,Ban K M,Guo Y F,Yang F L,Wei Y M.2010.Status of Fasciola gigantica infection in buffaloes in Qinzhou suburb of Guangxi[J].Guangxi Agricultural Sciences,41(11):1237-1239.]
黎万奎,陈幼竹,周宇,徐恒.2003.肝片吸虫抗原基因转基因苜蓿再生的研究[J].四川大学学报(自然科学版),40(1):144-147.[Li W K,Chen Y Z,Zhou Y,Xu H.2003.Regeneration of F.hepatica protective antigenic gene transgenic alfalfa plants by agrobacterium mediated gene transfer[J].Journal of Sichuan University(Natural Science Edition),40(1):144-147.]
李晓娟.2009.肝片吸虫重组GST、CatL蛋白及其DNA核酸联合免疫初步研究[D].大庆:黑龙江八一农垦大学.[Li XJ.2009.Preliminary study on combination immunization with recombinant proteins and DNA nucleicacids of GSTand CatL genes of Fasciola hepatica[D].Daqing:Heilongjiang Bayi Agricultural University.]
田艾灵,朱兴全,黄思扬.2017.片形吸虫排泄分泌产物的研究进展[J].畜牧兽医学报,48(2):201-206.[Tian A L,Zhu X Q,Huang S Y.2017.Research advance in excretory-secretory products of Fasciola spp[J].Acta Veterinaria et Zootechnica Sinica,48(2):201-206.]
王熙凤,孟庆玲,乔军,陈英,钟文强,贡莎莎,黄运福,才学鹏.2018.肝片吸虫Ftn-1蛋白分子特征、抗原性及其抗体制备[J].河南农业科学,47(2):119-124.[Wang X F,Meng Q L,Qiao J,Chen Y,Zhong W Q,Gong S S,Huang Y F,Cai X P.2018.Molecular characterization,antigenicity and antibody preparation of Ftn-1 protein of Fasciola hepatica[J].Journal of Henan Agricultural Sciences,47(2):119-124.]
Ashrafi K,Valero M A,Peixoto R V,Artigas P,Panova M,Mas-Coma S.2015.Distribution of Fasciola hepatica and F.gigantica in the endemic area of Guilan,Iran:Relationships between zonal overlap and phenotypic traits[J].Infection,Genetics and Evolution,31:95-109.
B?ska P,Norbury L J,Wi?niewski M,Januszkiewicz K,W?drychowicz H.2013.Excretory/secretory products of Fasciola hepatica but not recombinant phosphoglycerate kinase induce death of human hepatocyte cells[J].Acta Parasitologica,58(2):215-217.
Beckham S A,Piedrafita D,Phillips C I,Samarawickrema N,Law R H,Smooker P M,Quinsey N S,Irving J A,Greenwood D,Verhelst S H,Bogyo M,Turk B,Coetzer T H,Wijeyewickrema L C,Spithill T W,Pike R N.2009.Amajor cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites[J].The International Journal of Biochemistry&Cell Biology,41(7):1601-1612.
Cancela M,Acosta D,Rinaldi G,Silva E,Durán R,Roche L,Zaha A,Carmona C,Tort J F.2008.A distinctive repertoire of cathepsins is expressed by juvenile invasive Fasciola hepatica[J].Biochimie,90(10):1461-1475.
Cervi L,Rubinstein H,Masih D T.1996.Involvement of excretion-secretion products from Fasciola hepatica inducing suppression of the cellular immune responses[J].Veterinary Parasitology,61(1-2):97-111.
Chantree P,Phatsara M,Meemon K,Chaichanasak P,Changklungmoa N,Kueakhai P,Lorsuwannarat N,Sangpairoj K,Songkoomkrong S,Wanichanon C,Itagaki T,Sobhon P.2013.Vaccine potential of recombinant cathepsin Bagainst Fasciola gigantica[J].Experimental Parasitology,135(1):102-109.
Creaney J,Wilson L,Dosen M,Sandeman R M,Spithill T W,Parsons J C.1996.Fasciola hepatica:Irradiation-induced alterations in carbohydrate and cathepsin-B protease expression in newly excysted juvenile liver fluke[J].Experimental Parasitology,83(2):202-215.
Cwiklinski K,Dalton J P,Dufresne P J,La Course J,Williams D J,Hodgkinson J,Paterson S.2015.The Fasciola hepatica genome:Gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution[J].Genome Biology,16:71.doi:10.1186/s13059-015-0632-2.
Flynn R J,Mulcahy G.2008.Possible role for Toll-like receptors in interaction of Fasciola hepatica excretory/secretory products with bovine macrophages[J].Infection and Immunity,76(2):678-684.
Hillyer G V.2005.Fasciola antigens as vaccines against fascioliasis and schistosomiasis[J].Journal of Helminthology,79(3):241-247.
Jayaraj R,Piedrafita D,Dynon K,Grams R,Spithill T W,Smooker P M.2009.Vaccination against fasciolosis by a multivalent vaccine of stage-specific antigens[J].Veterinary Parasitology,160(3-4):230-236.
Kumar N,Anju V,Gaurav N,Chandra D,Samanta S,Gupta SC,Adeppa J,Raina O K.2012.Vaccination of buffaloes with Fasciola gigantica recombinant glutathione S-transferase and fatty acid binding protein[J].Parasitology Research,110(1):419-426.
López-Abán J,Casanueva P,Nogal J,Arias M,Morrondo P,Diez-Ba?os P,Hillyer G V,Martínez-Fernández A R,Muro A.2007.Progress in the development of Fasciola hepatica vaccine using recombinant fatty acid binding protein with the adjuvant adaptation system ADAD[J].Veterinary Parasitology,145(3-4):287-296.
Maggioli G,Silveira F,Martín-Alonso J M,Salinas G,Carmona C,Parra F.2011.A recombinant thioredoxin-glutathione reductase from Fasciola hepatica induces a protective response in rabbits[J].Experimental Parasitology,129(4):323-330.
Mas-Coma S,Valero M A,Bargues M D.2009.Chapter 2.Fasciola,lymnaeids and human fascioliasis,with a global overview on disease transmission,epidemiology,evolutionary genetics,molecular epidemiology and control[J].Advances in Parasitology,69:141-146.
Meemon K,Sobhon P.2015.Juvenile-specific cathepsin proteases in Fasciola spp.:Their characteristics and vaccine efficacies[J].Parasitology Research,114(8):2807-2813.
Robinson M W,Menon R,Donnelly S M,Dalton J P,Ranganathan S.2009.An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica:Proteins associated with invasion and infection of the mammalian host[J].Molecular&Cellular Proteomics,8(8):1891-1907.
Robinson M W,Tort J F,Lowther J,Donnelly S M,Wong E,Xu W,Stack C M,Padula M,Herbert B,Dalton J P.2008.Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica:Expansion of a repertoire of virulence-associated factors[J].Molecular&Cellular Proteomics,7(6):1111-1123.
Weso?owska A,Basa?aj K,Norbury L J,Sielicka A,W?drychowicz H,Zawistowska-Deniziak A.2018.Vaccination against Fasciola hepatica using cathepsin L3 and B3 proteases delivered alone or in combination[J].Veterinary Parasitology,250:15-21.doi:10.1016/j.vetpar.2017.12.007.
黎万奎,陈幼竹,周宇,徐恒.2003.肝片吸虫抗原基因转基因苜蓿再生的研究[J].四川大学学报(自然科学版),40(1):144-147.[Li W K,Chen Y Z,Zhou Y,Xu H.2003.Regeneration of F.hepatica protective antigenic gene transgenic alfalfa plants by agrobacterium mediated gene transfer[J].Journal of Sichuan University(Natural Science Edition),40(1):144-147.]
李晓娟.2009.肝片吸虫重组GST、CatL蛋白及其DNA核酸联合免疫初步研究[D].大庆:黑龙江八一农垦大学.[Li XJ.2009.Preliminary study on combination immunization with recombinant proteins and DNA nucleicacids of GSTand CatL genes of Fasciola hepatica[D].Daqing:Heilongjiang Bayi Agricultural University.]
田艾灵,朱兴全,黄思扬.2017.片形吸虫排泄分泌产物的研究进展[J].畜牧兽医学报,48(2):201-206.[Tian A L,Zhu X Q,Huang S Y.2017.Research advance in excretory-secretory products of Fasciola spp[J].Acta Veterinaria et Zootechnica Sinica,48(2):201-206.]
王熙凤,孟庆玲,乔军,陈英,钟文强,贡莎莎,黄运福,才学鹏.2018.肝片吸虫Ftn-1蛋白分子特征、抗原性及其抗体制备[J].河南农业科学,47(2):119-124.[Wang X F,Meng Q L,Qiao J,Chen Y,Zhong W Q,Gong S S,Huang Y F,Cai X P.2018.Molecular characterization,antigenicity and antibody preparation of Ftn-1 protein of Fasciola hepatica[J].Journal of Henan Agricultural Sciences,47(2):119-124.]
Ashrafi K,Valero M A,Peixoto R V,Artigas P,Panova M,Mas-Coma S.2015.Distribution of Fasciola hepatica and F.gigantica in the endemic area of Guilan,Iran:Relationships between zonal overlap and phenotypic traits[J].Infection,Genetics and Evolution,31:95-109.
B?ska P,Norbury L J,Wi?niewski M,Januszkiewicz K,W?drychowicz H.2013.Excretory/secretory products of Fasciola hepatica but not recombinant phosphoglycerate kinase induce death of human hepatocyte cells[J].Acta Parasitologica,58(2):215-217.
Beckham S A,Piedrafita D,Phillips C I,Samarawickrema N,Law R H,Smooker P M,Quinsey N S,Irving J A,Greenwood D,Verhelst S H,Bogyo M,Turk B,Coetzer T H,Wijeyewickrema L C,Spithill T W,Pike R N.2009.Amajor cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites[J].The International Journal of Biochemistry&Cell Biology,41(7):1601-1612.
Cancela M,Acosta D,Rinaldi G,Silva E,Durán R,Roche L,Zaha A,Carmona C,Tort J F.2008.A distinctive repertoire of cathepsins is expressed by juvenile invasive Fasciola hepatica[J].Biochimie,90(10):1461-1475.
Cervi L,Rubinstein H,Masih D T.1996.Involvement of excretion-secretion products from Fasciola hepatica inducing suppression of the cellular immune responses[J].Veterinary Parasitology,61(1-2):97-111.
Chantree P,Phatsara M,Meemon K,Chaichanasak P,Changklungmoa N,Kueakhai P,Lorsuwannarat N,Sangpairoj K,Songkoomkrong S,Wanichanon C,Itagaki T,Sobhon P.2013.Vaccine potential of recombinant cathepsin Bagainst Fasciola gigantica[J].Experimental Parasitology,135(1):102-109.
Creaney J,Wilson L,Dosen M,Sandeman R M,Spithill T W,Parsons J C.1996.Fasciola hepatica:Irradiation-induced alterations in carbohydrate and cathepsin-B protease expression in newly excysted juvenile liver fluke[J].Experimental Parasitology,83(2):202-215.
Cwiklinski K,Dalton J P,Dufresne P J,La Course J,Williams D J,Hodgkinson J,Paterson S.2015.The Fasciola hepatica genome:Gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution[J].Genome Biology,16:71.doi:10.1186/s13059-015-0632-2.
Flynn R J,Mulcahy G.2008.Possible role for Toll-like receptors in interaction of Fasciola hepatica excretory/secretory products with bovine macrophages[J].Infection and Immunity,76(2):678-684.
Hillyer G V.2005.Fasciola antigens as vaccines against fascioliasis and schistosomiasis[J].Journal of Helminthology,79(3):241-247.
Jayaraj R,Piedrafita D,Dynon K,Grams R,Spithill T W,Smooker P M.2009.Vaccination against fasciolosis by a multivalent vaccine of stage-specific antigens[J].Veterinary Parasitology,160(3-4):230-236.
Kumar N,Anju V,Gaurav N,Chandra D,Samanta S,Gupta SC,Adeppa J,Raina O K.2012.Vaccination of buffaloes with Fasciola gigantica recombinant glutathione S-transferase and fatty acid binding protein[J].Parasitology Research,110(1):419-426.
López-Abán J,Casanueva P,Nogal J,Arias M,Morrondo P,Diez-Ba?os P,Hillyer G V,Martínez-Fernández A R,Muro A.2007.Progress in the development of Fasciola hepatica vaccine using recombinant fatty acid binding protein with the adjuvant adaptation system ADAD[J].Veterinary Parasitology,145(3-4):287-296.
Maggioli G,Silveira F,Martín-Alonso J M,Salinas G,Carmona C,Parra F.2011.A recombinant thioredoxin-glutathione reductase from Fasciola hepatica induces a protective response in rabbits[J].Experimental Parasitology,129(4):323-330.
Mas-Coma S,Valero M A,Bargues M D.2009.Chapter 2.Fasciola,lymnaeids and human fascioliasis,with a global overview on disease transmission,epidemiology,evolutionary genetics,molecular epidemiology and control[J].Advances in Parasitology,69:141-146.
Meemon K,Sobhon P.2015.Juvenile-specific cathepsin proteases in Fasciola spp.:Their characteristics and vaccine efficacies[J].Parasitology Research,114(8):2807-2813.
Robinson M W,Menon R,Donnelly S M,Dalton J P,Ranganathan S.2009.An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica:Proteins associated with invasion and infection of the mammalian host[J].Molecular&Cellular Proteomics,8(8):1891-1907.
Robinson M W,Tort J F,Lowther J,Donnelly S M,Wong E,Xu W,Stack C M,Padula M,Herbert B,Dalton J P.2008.Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica:Expansion of a repertoire of virulence-associated factors[J].Molecular&Cellular Proteomics,7(6):1111-1123.
Weso?owska A,Basa?aj K,Norbury L J,Sielicka A,W?drychowicz H,Zawistowska-Deniziak A.2018.Vaccination against Fasciola hepatica using cathepsin L3 and B3 proteases delivered alone or in combination[J].Veterinary Parasitology,250:15-21.doi:10.1016/j.vetpar.2017.12.007.