大片吸虫钙结合蛋白2基因的克隆表达及其表达产物的免疫活性研究
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摘要
通过克隆、表达大片吸虫钙结合蛋白2(FgCaBP2)基因,旨在探索其作为诊断抗原的可行性。在分析大片吸虫蛋白组数据的基础上,根据肝片吸虫CaBP2基因组序列和转录组数据,设计以NdeⅠ和XhoⅠ作为酶切位点的引物。以大片吸虫总RNA为模板,通过RT-PCR获得FgCaBP2基因,对其进行测序、生物信息学分析。构建pET-30a-FgCaBP2重组表达载体,在大肠杆菌Transetta(DE3)感受态细胞中完成诱导、表达;对纯化的目的蛋白进行SDS-PAGE和Western-blot分析,并用目的蛋白免疫家兔,制备多克隆抗体。结果表明,FgCaBP2基因的开放阅读框长570 bp,编码189个氨基酸残基。生物信息学预测显示,FgCaBP2主要以α螺旋为主,β折叠较少,有7个β转角、4个较长的无规则卷曲、含有4个亲水区、具有较多的B细胞抗原表位。SDS-PAGE分析表明,FgCaBP2呈现出可溶性表达。Western-blot分析表明,25 ku大小的特异性目的蛋白条带能够很好地被大片吸虫感染的阳性水牛血清识别,具有良好的免疫反应性。本研究成功克隆了FgCaBP2基因,并获得了可溶性重组蛋白,该蛋白具有良好的免疫原性和免疫反应性,是潜在的大片吸虫病诊断抗原。
The objectives of the present study were to clone and express the gene of Fasciola gigantica calcium binding protein 2(FgCaBP2),and to explore the protein's potential as a diagnostic antigen.Based on the F.gigantica transcriptome data and the genomic sequence of Fasciola hepatica CaBP2,primers containing restriction sites for NdeⅠand XhoⅠwere designed.The FgCaBP2 gene was obtained by RT-PCR amplification using total RNA from F.gigantica as a template,and its identification and bioinformatics analysis were performed.The recombinant expression vector pET-30 a-FgCaBP2 was constructed,then induced and expressed in Escherichia coli Transetta(DE3) competent cells.The purified recombinant FgCaBP2 was purified and analyzed by SDS-PAGE and Western-blot.After the rabbits were immunized with the recombinant FgCaBP2,polyclonal antibody was prepared.The results showed that the ORF of FgCaBP2 was 570 bp in length,encoding 189 amino acid residues.FgCaBP2 had many α-helix,but only a fewβ-sheets,7 β-turns,and 4 longer irregular curls,4 hydrophilic regions and many B-cell epitopes.SDS-PAGE analyses indicated FgCaBP2 exhibited soluble expression.Western-blot results showed that the target protein had a specific band at 25 ku and had a good recognition effect with the positive sera from buffaloes infected with F.gigantica.In conclusion,the FgCaBP2 gene was cloned,and its expressed recombinant protein had good immuno-reactivity,indicating that FgCaBP 2 could be a potential diagnostic antigen.
The objectives of the present study were to clone and express the gene of Fasciola gigantica calcium binding protein 2(FgCaBP2),and to explore the protein's potential as a diagnostic antigen.Based on the F.gigantica transcriptome data and the genomic sequence of Fasciola hepatica CaBP2,primers containing restriction sites for NdeⅠand XhoⅠwere designed.The FgCaBP2 gene was obtained by RT-PCR amplification using total RNA from F.gigantica as a template,and its identification and bioinformatics analysis were performed.The recombinant expression vector pET-30 a-FgCaBP2 was constructed,then induced and expressed in Escherichia coli Transetta(DE3) competent cells.The purified recombinant FgCaBP2 was purified and analyzed by SDS-PAGE and Western-blot.After the rabbits were immunized with the recombinant FgCaBP2,polyclonal antibody was prepared.The results showed that the ORF of FgCaBP2 was 570 bp in length,encoding 189 amino acid residues.FgCaBP2 had many α-helix,but only a fewβ-sheets,7 β-turns,and 4 longer irregular curls,4 hydrophilic regions and many B-cell epitopes.SDS-PAGE analyses indicated FgCaBP2 exhibited soluble expression.Western-blot results showed that the target protein had a specific band at 25 ku and had a good recognition effect with the positive sera from buffaloes infected with F.gigantica.In conclusion,the FgCaBP2 gene was cloned,and its expressed recombinant protein had good immuno-reactivity,indicating that FgCaBP 2 could be a potential diagnostic antigen.
引文
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[17]岳东梅,王金磊,黄思扬,等.大片吸虫α-微管蛋白基因的克隆表达及免疫反应性的研究[J].中国兽医科学,2017,47(04):495-499.YUE Dong-mei,WANG Jin-lei,HUANG Si-yang,et al.Cloning,expression and immune-reactivity ofα-tubulin gene of Fasciola gigantica[J].Chinese Veterinary Science,2017,47(04):495-499.(in Chinese)
[18]SENAWONG G,LAHA T,LOUKAS A,et al.Cloning,expression,and characterization of a novel Opisthorchis viverrini calcium-binding EF-hand protein[J].Parasitology International,2012,61(1):94-100.
[19]XU J,REN Y,XU X,et al.Schistosoma japonicum tegumental protein 20.8,role in reproduction through its calcium binding ability[J].Parasitology Research,2014,113(2):491-497.
[20]HU S,LAW P K,LV Z,et al.Molecular characterization of a calcium-binding protein SjCa8 from Schistosoma japonicum[J].Parasitology Research,2008,103(5):1047-1053.
[21]SUBPIPATTANA P,GRAMS R,VICHASRI-GRAMS S.Analysis of a calcium-binding EF-hand protein family in Fasciola gigantica[J].Experimental Parasitology,2012,130(4):364-373.
[22]RUIZ DE EGUINO A D,MACH魱N A,CASAIS R,et al.Cloning and expression in Escherichia coli of a Fasciola hepatica gene encoding a calcium-binding protein[J].Molecular and Biochemical Parasitology,1999,101(1-2):13-21.
[23]THOMAS C M,TIMSON D J.A mysterious family of calcium-binding proteins from parasitic worms[J].Biochemical Society Transactions,2016,44(4):1005-1010.
[24]BANFORD S,DRYSDALE O,HOEY E M,et al.FhCaBP3:a Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains[J].Biochimie,2013,95(4):751-758.
[25]ORR R,KINKEAD R,NEWMAN R,et al.FhCaBP4:a Fasciola hepatica calcium-binding protein with EF-hand and dynein light chain domains[J].Parasitology Research,2012,111(4):1707-1713.
[26]CWIKLINSKI K,DALTON J P.Advances in Fasciola hepatica research using‘omics'technologies[J].International Journal for Parasitology,2018,48(5):321-331.
[27]NGUYEN T H,THOMAS C M,TIMSON D J,et al.Fasciola hepatica calcium-binding protein FhCaBP2:structure of the dynein light chain-like domain[J].Parasitology Research,2016,115(7):2879-2886.
[2]MEHMOOD K,ZHANG H,SABIR A J,et al.A review on epidemiology,global prevalence and economical losses of fasciolosis in ruminants[J].Microbial Pathogenesis,2017,109:253-262.
[3]MAS-COMA S,VALERO M A,BARGUES M D.Fascioliasis[J].Advances in Experimental Medicine and Biology,2014,766:77-114.
[4]MEEMON K,SOBHON P.Juvenile-specific cathepsin proteases in Fasciola spp.:their characteristics and vaccine efficacies[J].Parasitology Research,2015,114(8):2807-2813.
[5]CWIKLINSKI K,O'NEILL S M,DONNELLY S,et al.A prospective view of animal and human fasciolosis[J].Parasite Immunology,2016,38(9):558-568.
[6]CHEN J X,CHEN M X,AI L,et al.An outbreak of human Fascioliasis gigantica in Southwest China[J].Plos One,2013,8(8):e71520.
[7]SABOURIN E,ALDA P,V魣ZQUEZ A,et al.Impact of human activities on fasciolosis transmission[J].Trends in Parasitology,2018,34(10):891-903.
[8]CARNEVALE S,MALANDRINI J B,PANTANO M L,et al.Fasciola hepatica infection in humans:overcoming problems for the diagnosis[J].Acta Parasitologica,2016,61(4):776-783.
[9]THOMAS C M,TIMSON D J.FhCaBP2:a Fasciola hepatica calcium-binding protein with EF-hand and dynein light chain domains[J].Parasitology,2015,142(11):1375-1386.
[10]袁晓丹,王春仁,朱兴全.片形吸虫病的危害与防制[J/OL].中国动物传染病学报:1-5[2018-09-18].http://kns.cnki.net/kcms/detail/31.2031.S.20180915.1127.022.html.YUAN Xiao-dan,WANG Chun-ren,ZHU Xing-quan.Hazardousness and control measures of fascioliasis[J/OL].Chinese Journal of Animal Infectious Diseases:1-5[2018-09-18].http://kns.cnki.net/kcms/detail/31.2031.S.20180915.1127.022.html.(in Chinese)
[11]袁晓丹,黄思扬,田艾灵,等.片形吸虫分子检测技术的研究进展[J/OL].中国兽医科学,2019,49(01):85-90.YUAN Xiao-dan,HUANG Si-yang,TIAN Ai-ling,et al.Research advances in molecular detection technique for Fasciola[J/OL].Chinese Veterinary Science,2019,49(01):85-90.(in Chinese)
[12]濮珏彪,唐莉莉,殷岑楠,等.华支睾吸虫钙结合蛋白Cs16的重组表达及血清诊断应用的初步评价[J].中国寄生虫学与寄生虫病杂志,2018,36(03):275-279+285.PU Jue-biao,TANG Li-li,YIN Cen-nan,et al.Recombinant expression of calcium-binding protein from Clonorchis sinensis and preliminary evaluation of its application in immunodiagnosis[J].Chinese Journal of Parasitology and Parasitic Diseases,2018,36(03):275-279+285.(in Chinese)
[13]CWIKLINSKI K,JEWHURST H,MCVEIGH P,et al.Infection by the helminth parasite Fasciola hepatica requires rapid regulation of metabolic,virulence,and invasive factors to adjust to its mammalian host[J].Molecular and Cellular Proteomics,2018,17(4):792-809.
[14]AI L,DONG S J,ZHANG W Y,et al.Specific PCR-based assays for the identification of Fasciola species:their development,evaluation and potential usefulness in prevalence surveys[J].Annals of Tropical Medicine and Parasitology,2010,104(1):65-72.
[15]田艾灵,张念章,张芙恺,等.大片吸虫Ras家族蛋白基因的克隆表达及免疫反应性研究[J].中国兽医科学,2018,48(04):412-418.TIAN Ai-ling,ZHANG Nian-zhang,ZHANG Fu-kai,et al.Cloning,expression and immune-reactivity of Ras-related protein of Fasciola gigantica[J].Chinese Veterinary Science,2018,48(04):412-418.(in Chinese)
[16]岳东梅.大片形吸虫ES与宿主互作抗原的筛选、鉴定及分析[D].黑龙江八一农垦大学,2017.YUE Dong-mei.Screening,identification and analysis the antigens of Fasciola gigantica ES interacting with the host[D].Heilongjiang Bayi Agricultural University,2017.(in Chinese)
[17]岳东梅,王金磊,黄思扬,等.大片吸虫α-微管蛋白基因的克隆表达及免疫反应性的研究[J].中国兽医科学,2017,47(04):495-499.YUE Dong-mei,WANG Jin-lei,HUANG Si-yang,et al.Cloning,expression and immune-reactivity ofα-tubulin gene of Fasciola gigantica[J].Chinese Veterinary Science,2017,47(04):495-499.(in Chinese)
[18]SENAWONG G,LAHA T,LOUKAS A,et al.Cloning,expression,and characterization of a novel Opisthorchis viverrini calcium-binding EF-hand protein[J].Parasitology International,2012,61(1):94-100.
[19]XU J,REN Y,XU X,et al.Schistosoma japonicum tegumental protein 20.8,role in reproduction through its calcium binding ability[J].Parasitology Research,2014,113(2):491-497.
[20]HU S,LAW P K,LV Z,et al.Molecular characterization of a calcium-binding protein SjCa8 from Schistosoma japonicum[J].Parasitology Research,2008,103(5):1047-1053.
[21]SUBPIPATTANA P,GRAMS R,VICHASRI-GRAMS S.Analysis of a calcium-binding EF-hand protein family in Fasciola gigantica[J].Experimental Parasitology,2012,130(4):364-373.
[22]RUIZ DE EGUINO A D,MACH魱N A,CASAIS R,et al.Cloning and expression in Escherichia coli of a Fasciola hepatica gene encoding a calcium-binding protein[J].Molecular and Biochemical Parasitology,1999,101(1-2):13-21.
[23]THOMAS C M,TIMSON D J.A mysterious family of calcium-binding proteins from parasitic worms[J].Biochemical Society Transactions,2016,44(4):1005-1010.
[24]BANFORD S,DRYSDALE O,HOEY E M,et al.FhCaBP3:a Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains[J].Biochimie,2013,95(4):751-758.
[25]ORR R,KINKEAD R,NEWMAN R,et al.FhCaBP4:a Fasciola hepatica calcium-binding protein with EF-hand and dynein light chain domains[J].Parasitology Research,2012,111(4):1707-1713.
[26]CWIKLINSKI K,DALTON J P.Advances in Fasciola hepatica research using‘omics'technologies[J].International Journal for Parasitology,2018,48(5):321-331.
[27]NGUYEN T H,THOMAS C M,TIMSON D J,et al.Fasciola hepatica calcium-binding protein FhCaBP2:structure of the dynein light chain-like domain[J].Parasitology Research,2016,115(7):2879-2886.