肝片吸虫MeCatL-B融合基因的构建、表达及其间接ELISA检测方法的建立
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摘要
为分析组织蛋白酶的抗原性及评价其作为肝片吸虫(Fh) ELISA诊断抗原的潜力,本研究采用RT-PCR技术扩增FhCatL1D和CatB4基因的优势抗原表位集中区段,利用重叠延伸PCR技术融合CatL1D和CatB4基因片段获得约810 bp的Fh CatL-B融合基因(MeCatL-B),构建原核重组表达载体pET-MeCatL-B,并将其转化入BL21 (DE3)感受态细胞中诱导表达,经SDS-PAGE和western blot检测,结果显示表达的重组蛋白MeCatL-B (rMeCatL-B)约46 ku,可以被Fh阳性血清特异性识别。以纯化的rMeCatL-B为包被抗原,经优化各反应条件后建立检测Fh抗体的间接ELISA方法,该检测方法的特异性强、敏感性高、重复性好。利用该方法对97份绵羊临床血清样品进行检测,并与商品化FhELISA抗体试剂盒检测结果对比,结果显示二者符合率达95.88%。本研究为进一步研发Fh血清学诊断试剂盒奠定了前期基础。
To reveal the antigenicity of cathepsins and evaluate its potential value as diagnostic antigen for the detection of Fasciola hepatica via ELISA, the dominant epitope-rich regions of F.hepatica CatL1 D and CatB4 genes were amplified by RT-PCR, and fused them by overlap extension PCR to obtain the 810 bp CatL-B fusion gene(MeCatL-B). The prokaryotic expression recombinant plasmie pET-MeCatL-B was constructed and then transformed into BL21(DE3) competent cells for expression. The results of SDS-PAGE and western blot showed that the expressed recombinant MeCatL-B(rMeCatL-B) was about46 ku, which reacted with the positive sheep serum against F.hepatica. The purified reMeCatL-B was used as the coating antigen to develop an indirect ELISA assay for detecting F.hepatica antibody after optimization of reaction conditions. The specificity,sensitivity and reproducibility of the indirect ELISA were very high. In addition, a total of 97 serum samples were detected by the indirect ELISA, and the coincidence was 95.88 % between the established indirect ELISA and the commercial F.hepatica antibody ELISA kit, which laid the foundation for the development of F.hepatica serological diagnostic kit.
To reveal the antigenicity of cathepsins and evaluate its potential value as diagnostic antigen for the detection of Fasciola hepatica via ELISA, the dominant epitope-rich regions of F.hepatica CatL1 D and CatB4 genes were amplified by RT-PCR, and fused them by overlap extension PCR to obtain the 810 bp CatL-B fusion gene(MeCatL-B). The prokaryotic expression recombinant plasmie pET-MeCatL-B was constructed and then transformed into BL21(DE3) competent cells for expression. The results of SDS-PAGE and western blot showed that the expressed recombinant MeCatL-B(rMeCatL-B) was about46 ku, which reacted with the positive sheep serum against F.hepatica. The purified reMeCatL-B was used as the coating antigen to develop an indirect ELISA assay for detecting F.hepatica antibody after optimization of reaction conditions. The specificity,sensitivity and reproducibility of the indirect ELISA were very high. In addition, a total of 97 serum samples were detected by the indirect ELISA, and the coincidence was 95.88 % between the established indirect ELISA and the commercial F.hepatica antibody ELISA kit, which laid the foundation for the development of F.hepatica serological diagnostic kit.
引文
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[12]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.
[13]Mokhtarian K,Akhlaghi L,Meamar A R,et al.Serodiagnosis of fasciolosis by fast protein liquid chromatography fractionated excretory/secretory antigens[J].Parasitol Res,2016,115(8):2957-2965.
[14]Meemon K,Sobhon P.Juvenile-specific cathepsin proteases in Fasciola spp.:Their characteristics and vaccine efficacies[J].Parasitol Res,2015,114(8):2807-2813.
[15]黄维义.肝片吸虫病(fasciolasis)免疫学研究综述[J].中国兽医寄生虫病,1997,3:53-55.
[16]Salimi-Bejestani M R,McG arry J W,Felstead S,et al.Development of an antibody-detection ELISA for Fasciola hepatica and its evaluation against a commercially available test[J].Res Vet Sci,2005,78(2):177-181.
[17]Martínez-Sernández V,Perteguer M J,Hernández-González A,et al.Comparison of recombinant cathepsins L1,L2,and L5 as ELISA targets for serodiagnosis of bovine and ovine fascioliasis[J].Parasitol Res,2018,117(5):1521-1534.
[18]Wesoíowska A,Basaáaj K,Norbury L J,et al.Vaccination against Fasciola hepatica using cathepsin L3 and B3 proteases delivered alone or in combination[J].Vet Parasitol,2018,250:15-21.
[2]Dalton J P,O'Neill S,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.
[3]Boray J C.Experimental fascioliasis in Australia[J].Adv Parasitol,1969,7:195-210.
[4]Diab T M,Mansour H H,Mahmoud S S.Fasciola gigantica:Parasitological and scanning electron microscopy study of the in vitro effects of ivermectin and/or artemether[J].Exp Parasitol,2010,124(3):279-284.
[5]Elliott T P,Kelley J M,Rawlin G,et al.High prevalence of fasciolosis and evaluation of drug efficacy against Fasciola hepatica in dairy cattle in the Maffra and Bairnsdale districts of Gippsland,Victoria,Australia[J].Vet Parasitol,2015,209(1/2):117-124.
[6]Calvani N E D,George S D,Windsor P A,et al.Comparison of early detection of Fasciola hepatica in experimentally infected Merino sheep by real-time PCR,coproantigen ELISA and sedimentation[J].Vet Parasitol,2018,251:85-89.
[7]Abdolahi Khabisi S,Sarkari B,Moshfe A,et al.Production of monoclonal antibody against excretory-secretory atigen of Fasciola hepatica and evaluation of its efficacy in the diagnosis of fascioliasis[J].Monoclon Antib Immunodiagn Immunother,2017,36(1):8-14.
[8]Shrifi Y,Farahnak A,Golestani A,et al.Triclabendazole effect on protease enzyme activity in the excretory-secretory products of Fasciola hepatica in vitro[J].Iran J Parasitol,2014,9(1):107-113.
[9]Robinson M W,Menon R,Donnelly S M,et al.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].Mol Cell Proteomics,2009,8(8):1891-1907.
[10]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.
[11]Anuracpreeda P,Chawengkirttikul R,Sobhon P.Immunodiagnosis of Fasciola gigantic infection using monoclonal antibodybased Sandwich ELISA and immunochromatographic assay for detection of circulating cathepsin L1 protease[J].PLoS One,2016,11(1):e0145650.
[12]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.
[13]Mokhtarian K,Akhlaghi L,Meamar A R,et al.Serodiagnosis of fasciolosis by fast protein liquid chromatography fractionated excretory/secretory antigens[J].Parasitol Res,2016,115(8):2957-2965.
[14]Meemon K,Sobhon P.Juvenile-specific cathepsin proteases in Fasciola spp.:Their characteristics and vaccine efficacies[J].Parasitol Res,2015,114(8):2807-2813.
[15]黄维义.肝片吸虫病(fasciolasis)免疫学研究综述[J].中国兽医寄生虫病,1997,3:53-55.
[16]Salimi-Bejestani M R,McG arry J W,Felstead S,et al.Development of an antibody-detection ELISA for Fasciola hepatica and its evaluation against a commercially available test[J].Res Vet Sci,2005,78(2):177-181.
[17]Martínez-Sernández V,Perteguer M J,Hernández-González A,et al.Comparison of recombinant cathepsins L1,L2,and L5 as ELISA targets for serodiagnosis of bovine and ovine fascioliasis[J].Parasitol Res,2018,117(5):1521-1534.
[18]Wesoíowska A,Basaáaj K,Norbury L J,et al.Vaccination against Fasciola hepatica using cathepsin L3 and B3 proteases delivered alone or in combination[J].Vet Parasitol,2018,250:15-21.