肝片吸虫CatL1D蛋白的免疫原性研究
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摘要
【目的】研究绵羊肝片吸虫(Fasciola hepatica,Fh)CatL1D蛋白的分子特征和免疫原性。【方法】利用RT-PCR技术扩增出CatL1D基因,用相关软件分析其编码蛋白的生物学信息。将CatL1D基因克隆入表达载体pET-32a(+)中,构建原核表达载体pET-CatL1D,进行BamHⅠ/XhoⅠ双酶切和测序鉴定。将pET-CatL1D载体转化入大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达后进行SDS-PAGE和Western blot分析;以纯化后的重组蛋白CatL1D为抗原免疫小鼠,利用间接ELISA方法检测抗体效价,分析其免疫原性。【结果】成功克隆了CatL1D基因,该基因全长981 bp,编码326个氨基酸,其中第21-79位氨基酸为组织蛋白酶前肽抑制剂结构域,第108-321位氨基酸为木瓜蛋白酶家族半胱氨酸蛋白酶保守结构域;CatL1D蛋白含有6个酪蛋白激酶Ⅱ磷酸化位点、11个豆蔻酰化位点、1个蛋白激酶C磷酸化位点,含有10个优势抗原表位。成功构建了pET-CatL1D原核表达载体,诱导表达出CatL1D重组蛋白;SDS-PAGE和Western blot分析表明,重组蛋白CatL1D分子质量为51 ku,以包涵体的形式表达,具有较强的反应原性。间接ELISA法检测结果表明,重组蛋白CatL1D免疫小鼠血清抗体效价可达1∶102 400,证实其具有良好的免疫原性。【结论】Fh重组蛋白CatL1D具有较强的抗原性,有开发为早期诊断抗原和亚单位疫苗的潜力。
【Objective】 The molecular characteristics and immunogenicity of CatL1 D protein in Fasciola hepatica(Fh) were studied.【Method】 The CatL1D gene was amplified by RT-PCR and the biological information of its encoded protein was analyzed by online software.The CatL1D gene was cloned into the expression vector pET-32 a(+) to construct the prokaryotic expression vector pET-CatL1 D,and it was identified by BamH Ⅰ/XhoⅠ double enzyme digestion and sequencing.The pET-CatL1 D vector was transformed into E.coli BL21(DE3),the expression was induced by IPTG,and the expressed products were analyzed by SDS-PAGE and Western blot.The purified recombinant protein CatL1 D was used as antigen to immunize mice.The antibody titer was detected by indirect ELISA and the immunogenicity was analyzed.【Result】 The CatL1D gene was cloned successfully with 981 bp and encoding 326 amino acids.It had a cathepsin propeptide inhibitor domain at positions 21-79 and had a conserved papain family of cysteine protease domain at amino acids 108-321;The Cat1 D protein contains six casein kinase Ⅱ phosphorylation sites,eleven myristoylation sites and a protein kinase C phosphorylation site;also containing ten dominant antigen epitopes.The pET-CatL1 D prokaryotic expression vector was successfully constructed and the expression of CatL1 D recombinant protein was induced.SDS-PAGE and Western blot analysis showed that the molecular weight of recombinant protein CatL1 D was 51 ku.It expressed as inclusion body and had good reactivity.The indirect ELISA result showed that the titer of serum antibody of mice immunized with recombinant protein CatL1 D was 1∶102 400,which proved its good immuoogeneticity.【Conclusion】 The Fh CatL1 D recombinant protein is highly antigenic and has potential for the development of early diagnostic antigens and subunit vaccines.
【Objective】 The molecular characteristics and immunogenicity of CatL1 D protein in Fasciola hepatica(Fh) were studied.【Method】 The CatL1D gene was amplified by RT-PCR and the biological information of its encoded protein was analyzed by online software.The CatL1D gene was cloned into the expression vector pET-32 a(+) to construct the prokaryotic expression vector pET-CatL1 D,and it was identified by BamH Ⅰ/XhoⅠ double enzyme digestion and sequencing.The pET-CatL1 D vector was transformed into E.coli BL21(DE3),the expression was induced by IPTG,and the expressed products were analyzed by SDS-PAGE and Western blot.The purified recombinant protein CatL1 D was used as antigen to immunize mice.The antibody titer was detected by indirect ELISA and the immunogenicity was analyzed.【Result】 The CatL1D gene was cloned successfully with 981 bp and encoding 326 amino acids.It had a cathepsin propeptide inhibitor domain at positions 21-79 and had a conserved papain family of cysteine protease domain at amino acids 108-321;The Cat1 D protein contains six casein kinase Ⅱ phosphorylation sites,eleven myristoylation sites and a protein kinase C phosphorylation site;also containing ten dominant antigen epitopes.The pET-CatL1 D prokaryotic expression vector was successfully constructed and the expression of CatL1 D recombinant protein was induced.SDS-PAGE and Western blot analysis showed that the molecular weight of recombinant protein CatL1 D was 51 ku.It expressed as inclusion body and had good reactivity.The indirect ELISA result showed that the titer of serum antibody of mice immunized with recombinant protein CatL1 D was 1∶102 400,which proved its good immuoogeneticity.【Conclusion】 The Fh CatL1 D recombinant protein is highly antigenic and has potential for the development of early diagnostic antigens and subunit vaccines.
引文
[1] Charlier J,Vercruysse J,Morgan E,et al.Recent advances in the diagnosis,impact on production and prediction of Fasciola hepatica in cattle [J].Parasitology,2014,141(3):326-335.
[2] Collins P R,Stack C M,O″Neill,et al.Cathepsin L1,the major protease involved in liver fluke (Fasciola hepatica) virulence:propetide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells [J].J Biol Chem,2004,79(17):17038-17046.
[3] Moll L,Gaasenbeek C P,Vellema P,et al.Resistance of Fasciola hepatica against triclabendazole in cattle and sheep in the Netherlands [J].Vet Parasitol,2000,91(1/2):153-158.
[4] Charlier J,Soenen K,De Roeck E,et al.Longitudinal study on the temporal and micro-spatial distribution of Galba truncatula in four farms in Belgium as a base for small-scale risk mapping of Fasciola hepatica [J].Parasit Vectors,2014,7:528.
[5] 周文茂.探究羊肝片吸虫病的流行病学特性及综合防治 [J].中国动物保健,2017,19(6):82-84. Zhou W M.Exploring the epidemiological characteristics and integrated prevention of fascioliasis in sheep [J].China Animal Health,2017,19(6):82-84.
[6] Robinson M W,Tort J F,Lowther J,et al.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].Mol Cell Proteomics,2008,7(6):1111-1123.
[7] Wilson R A,Wright J M,Dowle A A,et al.Exploring the Fasciola hepatica tegument proteome [J].Int J Parasitol,2011,41(13/14):1347-1359.
[8] Dalton J P,Robinson M W,Mulcahy G,et al.Immunomodulatory molecules of Fasciola hepatica:candidates for both vaccine and immunotherapeutic development [J].Vet Parasitol,2013,195(3/4):272-285.
[9] Prowse R K,Chaplin P,Robinson H C,et al.Fasciola hepatica cathepsin L suppresses sheep lymphocyte proliferation in vitro and modulates surface CD4 expression on human and ovine T cell [J].Parasite Immunol,2002,24(2):57-66.
[10] Khaznadji E,Collins P,Dalton J P,et al.A new multi-domain member of the cystatin superfamily expressed by Fasciola hepatica [J].Int J Parasitol,2005,35(10):1115-1125.
[11] 李润乐.新型肝片吸虫多表位疫苗的研究 [D].西宁:青海大学,2015. Li R L.Studies on a novel mulitiple-epitope vaccine against Fasciola hepatica [D].Xining:Qinghai University,2015.
[12] 徐怀英,朱瑞良,赵宏坤.肝片吸虫病诊断与防治研究进展 [J].中国兽医寄生虫病,2000(4):49-51. Xu H Y,Zhu R L,Zhao H K.Progress in diagnosis and control of fascioliasis [J].Chinese Journal of Veterinary Parasitology,2000(4):49-51.
[13] Kelley J M,Elliott T P,Beddoe T,et al.Current threat of triclabendazole resistance in Fasciola hepatica [J].Trends Parasitol,2016,32(6):458-469.
[14] Charlier J,Vercruysse J,Morgan E,et al.Recent advances in the diagnosis,impact on production and prediction of Fasciola hepatica in cattle [J].Parasitology,2014,141:326-335.
[15] Piedrafita D,Parsons J C,Sandeman R M,et al.Antibody-dependent cell-mediated cytotoxicity to newly excysted juvenile Fasciola hepatica in vitro is mediated by reactive nitrogen intermediates [J].Parasite Immunol,2001,23(9):473-482.
[16] Moreau E,Hervé S,Yu Z W,et al.Modulation of sheep lymphocyte responses by Fasciola hepatica excretory-secretory products [J].Vet Parasitol,2002,108(3):207-215.
[17] Huang S Y,Liu Q,Yue D M,et al.Proteomic analysis of Fasciola hepatica excretory and secretory products (FhESPs) involved in interacting with host PBMCs and cytokines by shotgun LC-MS/MS [J].Parasitol Res,2017,116(2):627-635.
[18] Dalton J P,Neill S O,Stack C,et al.Fasciola hepatica cathepsin L-like proteases:biology,function,and potential in the development of first generation liver fluke vaccines [J].Int J Parasitol,2003,33(11):1173-1181.
[19] Claridge J,Diggle P,McCann C,et al.Fasciola hepatica is associated with the failure to detect bovine tuberculosis in dairy cattle [J].Nat Commun,2012,3:853.
[20] Toet H,Piedrafita D M,Spithill T W.Liver fluke vaccines in ruminants:strategies,progress and future opportunities [J].Int J Parasitol,2014,44(12):915-927.
[21] Molina-Hernández V,Mulcahy G,Pérez J,et al.Fasciola hepatica vaccine:we may not be there yet but we're on the right road [J].Vet Parasitol,2015,208(1/2):101-111.
[22] Piacenza L,Acosta D,Basmadjian I,et al.Vaccination with cathepsin L proteinases and with leucine aminopeptidase induces high levels of protection against fascioliasis in sheep [J].Infect Immun,1999,67(4):1954-1961.
[23] Dalton J P,McGonigle S,Rolph T P,et al.Induction of protective immunity in cattle against infection with Fasciola hepatica by vaccination with cathepsin L proteinases and with hemoglobin [J].Infect Immun,1996,64(12):5066-5074.
[24] Mokhtarian K,Akhlaghi L,Mohammadi M,et al.Evaluation of anti-Cathepsin L1:a more reliable method for serodiagnosis of human fasciolosis [J].Trans R Soc Trop Med Hyg,2016,110(9):542-550.
[2] Collins P R,Stack C M,O″Neill,et al.Cathepsin L1,the major protease involved in liver fluke (Fasciola hepatica) virulence:propetide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells [J].J Biol Chem,2004,79(17):17038-17046.
[3] Moll L,Gaasenbeek C P,Vellema P,et al.Resistance of Fasciola hepatica against triclabendazole in cattle and sheep in the Netherlands [J].Vet Parasitol,2000,91(1/2):153-158.
[4] Charlier J,Soenen K,De Roeck E,et al.Longitudinal study on the temporal and micro-spatial distribution of Galba truncatula in four farms in Belgium as a base for small-scale risk mapping of Fasciola hepatica [J].Parasit Vectors,2014,7:528.
[5] 周文茂.探究羊肝片吸虫病的流行病学特性及综合防治 [J].中国动物保健,2017,19(6):82-84. Zhou W M.Exploring the epidemiological characteristics and integrated prevention of fascioliasis in sheep [J].China Animal Health,2017,19(6):82-84.
[6] Robinson M W,Tort J F,Lowther J,et al.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].Mol Cell Proteomics,2008,7(6):1111-1123.
[7] Wilson R A,Wright J M,Dowle A A,et al.Exploring the Fasciola hepatica tegument proteome [J].Int J Parasitol,2011,41(13/14):1347-1359.
[8] Dalton J P,Robinson M W,Mulcahy G,et al.Immunomodulatory molecules of Fasciola hepatica:candidates for both vaccine and immunotherapeutic development [J].Vet Parasitol,2013,195(3/4):272-285.
[9] Prowse R K,Chaplin P,Robinson H C,et al.Fasciola hepatica cathepsin L suppresses sheep lymphocyte proliferation in vitro and modulates surface CD4 expression on human and ovine T cell [J].Parasite Immunol,2002,24(2):57-66.
[10] Khaznadji E,Collins P,Dalton J P,et al.A new multi-domain member of the cystatin superfamily expressed by Fasciola hepatica [J].Int J Parasitol,2005,35(10):1115-1125.
[11] 李润乐.新型肝片吸虫多表位疫苗的研究 [D].西宁:青海大学,2015. Li R L.Studies on a novel mulitiple-epitope vaccine against Fasciola hepatica [D].Xining:Qinghai University,2015.
[12] 徐怀英,朱瑞良,赵宏坤.肝片吸虫病诊断与防治研究进展 [J].中国兽医寄生虫病,2000(4):49-51. Xu H Y,Zhu R L,Zhao H K.Progress in diagnosis and control of fascioliasis [J].Chinese Journal of Veterinary Parasitology,2000(4):49-51.
[13] Kelley J M,Elliott T P,Beddoe T,et al.Current threat of triclabendazole resistance in Fasciola hepatica [J].Trends Parasitol,2016,32(6):458-469.
[14] Charlier J,Vercruysse J,Morgan E,et al.Recent advances in the diagnosis,impact on production and prediction of Fasciola hepatica in cattle [J].Parasitology,2014,141:326-335.
[15] Piedrafita D,Parsons J C,Sandeman R M,et al.Antibody-dependent cell-mediated cytotoxicity to newly excysted juvenile Fasciola hepatica in vitro is mediated by reactive nitrogen intermediates [J].Parasite Immunol,2001,23(9):473-482.
[16] Moreau E,Hervé S,Yu Z W,et al.Modulation of sheep lymphocyte responses by Fasciola hepatica excretory-secretory products [J].Vet Parasitol,2002,108(3):207-215.
[17] Huang S Y,Liu Q,Yue D M,et al.Proteomic analysis of Fasciola hepatica excretory and secretory products (FhESPs) involved in interacting with host PBMCs and cytokines by shotgun LC-MS/MS [J].Parasitol Res,2017,116(2):627-635.
[18] Dalton J P,Neill S O,Stack C,et al.Fasciola hepatica cathepsin L-like proteases:biology,function,and potential in the development of first generation liver fluke vaccines [J].Int J Parasitol,2003,33(11):1173-1181.
[19] Claridge J,Diggle P,McCann C,et al.Fasciola hepatica is associated with the failure to detect bovine tuberculosis in dairy cattle [J].Nat Commun,2012,3:853.
[20] Toet H,Piedrafita D M,Spithill T W.Liver fluke vaccines in ruminants:strategies,progress and future opportunities [J].Int J Parasitol,2014,44(12):915-927.
[21] Molina-Hernández V,Mulcahy G,Pérez J,et al.Fasciola hepatica vaccine:we may not be there yet but we're on the right road [J].Vet Parasitol,2015,208(1/2):101-111.
[22] Piacenza L,Acosta D,Basmadjian I,et al.Vaccination with cathepsin L proteinases and with leucine aminopeptidase induces high levels of protection against fascioliasis in sheep [J].Infect Immun,1999,67(4):1954-1961.
[23] Dalton J P,McGonigle S,Rolph T P,et al.Induction of protective immunity in cattle against infection with Fasciola hepatica by vaccination with cathepsin L proteinases and with hemoglobin [J].Infect Immun,1996,64(12):5066-5074.
[24] Mokhtarian K,Akhlaghi L,Mohammadi M,et al.Evaluation of anti-Cathepsin L1:a more reliable method for serodiagnosis of human fasciolosis [J].Trans R Soc Trop Med Hyg,2016,110(9):542-550.
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