基于重组IBDV VP3蛋白的免疫磁珠间接ELISA抗体检测方法的建立及初步应用
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摘要
本研究旨在毕赤酵母中表达鸡传染性法氏囊病毒(infectious bursal disease virus,IBDV)的VP3蛋白,以重组VP3蛋白作为包被抗原建立IBDV血清抗体免疫磁珠间接ELISA检测方法并进行初步应用。利用毕赤酵母表达系统获得约为39 ku的重组VP3蛋白,Western-blotting检测表明,重组VP3蛋白具有良好的特异性和反应原性。免疫磁珠间接ELISA的最佳反应条件:磁珠和抗原偶联的缓冲液是50 mmol/L MES(p H 8.0),偶联时间为3 h,抗原加入量为95μg,抗原与羧基磁珠最大偶联量为80μg。待检血清和酶标二抗的稀释度分别为1∶1 600和1∶4 000,血清孵育时间为30 min,酶标二抗孵育时间为20 min。在该优化条件下,阴性、阳性临界值判定标准为0.134。特异性试验显示,对抗AIV、IBV、MDV、NDV阳性血清检测结果均为阴性。敏感性试验显示,本试验所建立的检测方法敏感性高于商品化IBDV血清抗体检测试剂盒。重复性试验显示,批内和批间重复试验的最大变异系数分别为3.8%和5.9%。稳定性试验结果显示变异系数小于7%。用建立的检测方法和商品化IBDV血清抗体检测试剂盒同时检测50份血清,结果显示两种检测方法的相对敏感性为94.6%,相对特异性为92.3%,符合率为94.0%。本试验建立的检测方法具有敏感性高、特异性强、重复性好、稳定性高特点,为其应用于临床IBDV血清抗体的检测奠定了基础。
This study aimed to express the VP3 protein of infectious bursal disease virus(IBDV) in Pichia pastoris to construct the immunomagnetic bead indirect ELISA for the detection of antibodies against the IBDV VP3 protein,with the recombinant VP3 protein as the coating antigen,and to carry out preliminary application. The recombinant VP3 protein about 39 ku in mass was obtained using the Pichia pastoris expression system,and Western-blotting detection suggested that the recombinant VP3 protein had possessed excellent specificity and immunoreactivity. The optimal reaction conditions for the immunomagnetic bead indirect ELISA were as follows:the coupling buffer solution for magnetic bead and antigen was 50 mmol/L MES(pH=8.0),the coupling time was 3 h,the antigen addition was 95μ g,and the maximum coupling amount of antigen with carboxyl magnetic bead was 80μ g.The serum and conjugated reagent were diluted at the ratios of 1 ∶ 1 600 and 1 ∶ 4 000,respectively,the serum incubation time was30 min,and the incubation time of conjugated secondary antibody was 20 min.Under those optimization conditions,the criterion for negative and positive thresholds was 0.134.The specificity test suggested that,all test results of positive serum anti-AIV,anti-IBV,anti-MDV and anti-NDV were negative.The sensitivity test suggested that,the detection method constructed in this experiment had higher sensitivity than the commercial IBDV serum antibody detection kit. The repeatability test indicated that,the maximum variation coefficients of intra-batch and inter-batch repeatability tests were 3.8% and5.9%,respectively.The stability test results demonstrated that,the variation coefficient was smaller than 7%.Subsequently,the constructed detection method as well as the commercial IBDV serum antibody detection kit was used to detect 50 serum samples at the same time,and the results suggested that,the relative sensitivity of these two detection methods was 94.0%,the relative specificity was92.3%,and the coincidence rate was 97.6%.Obviously,the detection method constructed in this experiment has high sensitivity,high specificity,good repeatability and high stability,which lay the foundation for its clinical application in detecting IBDV serum antibodies.
This study aimed to express the VP3 protein of infectious bursal disease virus(IBDV) in Pichia pastoris to construct the immunomagnetic bead indirect ELISA for the detection of antibodies against the IBDV VP3 protein,with the recombinant VP3 protein as the coating antigen,and to carry out preliminary application. The recombinant VP3 protein about 39 ku in mass was obtained using the Pichia pastoris expression system,and Western-blotting detection suggested that the recombinant VP3 protein had possessed excellent specificity and immunoreactivity. The optimal reaction conditions for the immunomagnetic bead indirect ELISA were as follows:the coupling buffer solution for magnetic bead and antigen was 50 mmol/L MES(pH=8.0),the coupling time was 3 h,the antigen addition was 95μ g,and the maximum coupling amount of antigen with carboxyl magnetic bead was 80μ g.The serum and conjugated reagent were diluted at the ratios of 1 ∶ 1 600 and 1 ∶ 4 000,respectively,the serum incubation time was30 min,and the incubation time of conjugated secondary antibody was 20 min.Under those optimization conditions,the criterion for negative and positive thresholds was 0.134.The specificity test suggested that,all test results of positive serum anti-AIV,anti-IBV,anti-MDV and anti-NDV were negative.The sensitivity test suggested that,the detection method constructed in this experiment had higher sensitivity than the commercial IBDV serum antibody detection kit. The repeatability test indicated that,the maximum variation coefficients of intra-batch and inter-batch repeatability tests were 3.8% and5.9%,respectively.The stability test results demonstrated that,the variation coefficient was smaller than 7%.Subsequently,the constructed detection method as well as the commercial IBDV serum antibody detection kit was used to detect 50 serum samples at the same time,and the results suggested that,the relative sensitivity of these two detection methods was 94.0%,the relative specificity was92.3%,and the coincidence rate was 97.6%.Obviously,the detection method constructed in this experiment has high sensitivity,high specificity,good repeatability and high stability,which lay the foundation for its clinical application in detecting IBDV serum antibodies.
引文
[1]ALFONSO A,NODA J.Feasibility of using IBDV VP3 recombinant protein in an immunosorbent assay(ELISA)[J].Redvet,2010,11(6):061002.
[2]廖洁丹,陈玉明,张济培.鸡传染性法氏囊病病毒的分子致病机理研究进展[J].中国家禽,2018,40(5):1-5.LIAO Jie-dan,CHEN Yu-ming,ZHANG Ji-pei.Research progress on molecular pathogenesis of infectious bursal disease virus[J].Chinese Poultry,2018,40(5):1-5.(in Chinese)
[3]WANG M Y,HU H L,SUN S Y,et al.Development of an enzyme-linked immunosorbent assay for detecting infectious bursal disease virus(IBDV)infection based on the VP3 structural protein[J].Veterinary Microbiology,2008,131(3):621-627.
[4]宋铁彬,陈广东.利用VP3蛋白初步建立鸡传染性法氏囊病间接ELISA方法[J].中国畜牧兽医,2011,38(1):116-118.SONG Tie-bin,CHEN Guang-dong.Preliminary establishment of indirect ELISA based on the VP3 structural protein of IBDV[J].China Animal Husbandry&Veterinary Medicine,2011,38(1):116-118.(in Chinese)
[5]MAT AS R,EVANGELINA G,MARI A S L,et al.Comparison of homologous and heterologous prime-boost immunizations combining MVA-vectored and plantderived VP2 as a strategy against IBDV[J].Vaccine,2017,35(1):142-148.
[6]LIU L L,ZHANG W,SONG Y X,et al.Recombinant Lactococcus lactis co-expressing OmpH of an M cell-targeting ligand and IBDV-VP2 protein provide immunological protection in chickens[J].Vaccine,2018,36(5):328-330.
[7]张华,南文龙,巩明霞,等.鸡传染性法氏囊病血清抗体间接ELISA检测方法的建立[J].中国动物检疫,2015,32(9):72-76.ZHANG Hua,NAN Wen-long,GONG Ming-xia,et al.Development of an indirect ELISA for the detection of antibodies against infectious bursal disease virus in chickens[J].Chinese Animal Quarantine,2015,32(9):72-76.(in Chinese)
[8]SHAW I,DAVISON T F.Protection from IBDV-induced bursal damage by a recombinant fowlpox vaccine,fpIBD1,is dependent on the titre of challenge virus and chicken genotype[J].Vaccine,2000,18(28):3230-3241.
[9]谢芳,赖卫华,史爱武,等.免疫磁珠富集结合酶联免疫吸附法检测酱油中黄曲霉毒素B1[J].食品科学,2013,34(18):165-169.XIE Fang,LAI Wei-hua,SHI Ai-wu,et al.Immunomagnetic bead enrichment and ELISA for detection of aflatoxin B1 in soy sauce by immunomagnetic beads enrichment and enzyme linked immunosorbent assay(ELISA)[J].Food Science,2013,34(18):165-169.(in Chinese)
[10]吴朝金.凡纳滨对虾体内隐蔽态T-2毒素的共性表征与危害识别[D].湛江:广东海洋大学,2014:68-83.WU Chao-jin.The common representation and hazard identification of masked T-2 toxin in litopenaeus vannamei[D].Zhanjiang:Guangdong Ocean University,2014:68-83.(in Chinese)
[11]胡泽宇.苊单克隆抗体的制备及免疫磁珠-ELISA方法的建立[D].长春:吉林大学,2016:25-28.HU Ze-yu.Preparation of monoclonal antibody and development of an immunoassay based on immunomagnetic bead for the acenaphthene[D].Changchun:Jilin University,2016:25-28.(in Chinese)
[12]马秀丽,崔言顺,张秀美,等.鸡传染性法氏囊病间接ELISA诊断试剂盒的研制及初步应用[J].中国兽医学报,2003,23(5):424-426.MA Xiu-li,CUI Yan-shun,ZHANG Xiu-mei,et al.Development and validation of indirect en zyme-linked immunosorbent assay test kit for detection of IBDV antibody[J].Journal of Veterinary Medicine,2003,23(5):424-426.(in Chinese)
[13]郭慧芳.基于可重复使用免疫磁珠的抗体检测方法建立及可重复使用磁性微流体蛋白芯片初步设计[D].西安:第四军医大学,2006:5-9.GUO Hui-fang.A new method to detect antibodies based on immunomagnetic beads that can be used to repeatedly and the primary frame of the micro-hydromagnet proteinic-chip that can be used repeatedly[D].Xi’an:Fourth military Medical University,2006:5-9.(in Chinese)
[14]刘华洁,杜希宁,梅永杰,等.传染性法氏囊病病毒VP3基因在昆虫细胞中的表达及其ELISA抗原反应性的分析[J].中国兽医科学,2015,45(3):227-234.LIU Hua-jie,DU Xi-ning,MEI Yong-jie,et al.Expression of VP3 gene of infectious bural disease virus in insect cells and analysis of antigenicity for VP3-ELISA[J].Chinese Veterinary Science,2015,45(3):227-234.(in Chinese)
[15]刘婷.IBDV主要结构蛋白VP2、VP1原核表达及其ELISA抗体检测试剂盒的研发[D].南宁:广西大学,2016:53-58.LIU Ting.Prokaryotic expression of IBDV major structural protein VP2,VP1 and development of ELISA antibody detection kit[D].Nanning:Guangxi University,2016:53-58.(in Chinese)
[16]SHIRVANI E,PALDURAI A,MANOHARAN V K,et al.A recombinant newcastle disease virus(NDV)expressing protein of infectious bronchitis virus(IBV)protects chickens against IBV and NDV[J].Scientific Reports,2018(8):521-527.
[17]IRAM M,SHI W,WANG L,et al.Immunogenicity and protective efficacy of orally administered recombinant Lactobacillus plantarum expressing VP2 protein against IBDV in chicken[J].Journal of Applied Microbiology,2018,32(3):6-8.
[18]宋军,黄成斌,单雪芹,等.传染性法氏囊病毒VP2蛋白的表达及间接ELISA抗体检测方法的建立[J].安徽农业大学学报,2011,38(4):633-636.SONG Jun,HUANG Cheng-bin,SHAN Xue-qin,et al.Establishment of an indirect ELISA method using a infectious bursal disease virus VP2 protein as antigen[J].Journal of Anhui Agricultural University,2011,38(4):633-636.(in Chinese)
[19]王晓丽,诸玉梅,夏兴霞,等.传染性法氏囊病病毒VP2单克隆抗体夹心ELISA检测试剂盒的研制[J].中国兽医学报,2014,34(4):530-535.WANG Xiao-li,ZHU Yu-mei,XIA Xing-xia,et al.Development of sandwich ELISA Kit for detection of infectious bursal disease virus VP2 monoclonal antibody[J].Chinese Journal of Veterinary Medicine,2014,34(4):530-535.(in Chinese)
[20]SINGH N K,DEY S,MADHAN M C,et al.Evaluation of four enzyme linked immunosorbent assays for the detection of antibodies to infectious bursal disease in chickens[J].Journal of Virological Methods,2010,165(2):277-282.
[21]FAHEY K J,O'DONNELL I J,AZAD A A.Characterization by Western-blotting of the immunogens of infectious bursal disease virus[J].Journal of General Virology,1985,66(Pt7):1479-1488.
[22]王慧宁.IBDV VP2-VP5-IgY Fc融合蛋白在毕赤酵母的表达及其免疫原性的研究[D].泰安:山东农业大学,2018:2-6.WANG Hui-ning.Expression and immunogenicity of IBDV VP2-VP5-IgY Fc fusion protein in Pichia pastoris[D].Taian:Shandong Agricultural University,2018:2-6.(in Chinese)
[23]王晓丽,王永山,欧阳伟,等.传染性法氏囊病病毒VP2结构蛋白抗体ELISA检测方法的建立[J].中国兽医学报,2018,38(10):1864-1871.WANG Xiao-li,WANG Yong-shan,OUYANG Wei,et al.Establishment of an ELISA method for the detection of IBDV antibody by using the viral specific VP2 structural proteins[J].Journal of Veterinary Medicine,2018,38(10):1864-1871.(in Chinese)
[24]郑玲燕.传染性法氏囊病毒VP2基因的克隆、表达及ELISA抗体检测方法的建立[D].武汉:华中农业大学,2006:123-127.ZHENG Ling-yan.Cloning and expression of VP2gene of infectious bursal virus and preliminary development of ELISA antibody detecting[D].Wuhan:Huazhong Agricultural University,2006:123-127.(in Chinese)
[2]廖洁丹,陈玉明,张济培.鸡传染性法氏囊病病毒的分子致病机理研究进展[J].中国家禽,2018,40(5):1-5.LIAO Jie-dan,CHEN Yu-ming,ZHANG Ji-pei.Research progress on molecular pathogenesis of infectious bursal disease virus[J].Chinese Poultry,2018,40(5):1-5.(in Chinese)
[3]WANG M Y,HU H L,SUN S Y,et al.Development of an enzyme-linked immunosorbent assay for detecting infectious bursal disease virus(IBDV)infection based on the VP3 structural protein[J].Veterinary Microbiology,2008,131(3):621-627.
[4]宋铁彬,陈广东.利用VP3蛋白初步建立鸡传染性法氏囊病间接ELISA方法[J].中国畜牧兽医,2011,38(1):116-118.SONG Tie-bin,CHEN Guang-dong.Preliminary establishment of indirect ELISA based on the VP3 structural protein of IBDV[J].China Animal Husbandry&Veterinary Medicine,2011,38(1):116-118.(in Chinese)
[5]MAT AS R,EVANGELINA G,MARI A S L,et al.Comparison of homologous and heterologous prime-boost immunizations combining MVA-vectored and plantderived VP2 as a strategy against IBDV[J].Vaccine,2017,35(1):142-148.
[6]LIU L L,ZHANG W,SONG Y X,et al.Recombinant Lactococcus lactis co-expressing OmpH of an M cell-targeting ligand and IBDV-VP2 protein provide immunological protection in chickens[J].Vaccine,2018,36(5):328-330.
[7]张华,南文龙,巩明霞,等.鸡传染性法氏囊病血清抗体间接ELISA检测方法的建立[J].中国动物检疫,2015,32(9):72-76.ZHANG Hua,NAN Wen-long,GONG Ming-xia,et al.Development of an indirect ELISA for the detection of antibodies against infectious bursal disease virus in chickens[J].Chinese Animal Quarantine,2015,32(9):72-76.(in Chinese)
[8]SHAW I,DAVISON T F.Protection from IBDV-induced bursal damage by a recombinant fowlpox vaccine,fpIBD1,is dependent on the titre of challenge virus and chicken genotype[J].Vaccine,2000,18(28):3230-3241.
[9]谢芳,赖卫华,史爱武,等.免疫磁珠富集结合酶联免疫吸附法检测酱油中黄曲霉毒素B1[J].食品科学,2013,34(18):165-169.XIE Fang,LAI Wei-hua,SHI Ai-wu,et al.Immunomagnetic bead enrichment and ELISA for detection of aflatoxin B1 in soy sauce by immunomagnetic beads enrichment and enzyme linked immunosorbent assay(ELISA)[J].Food Science,2013,34(18):165-169.(in Chinese)
[10]吴朝金.凡纳滨对虾体内隐蔽态T-2毒素的共性表征与危害识别[D].湛江:广东海洋大学,2014:68-83.WU Chao-jin.The common representation and hazard identification of masked T-2 toxin in litopenaeus vannamei[D].Zhanjiang:Guangdong Ocean University,2014:68-83.(in Chinese)
[11]胡泽宇.苊单克隆抗体的制备及免疫磁珠-ELISA方法的建立[D].长春:吉林大学,2016:25-28.HU Ze-yu.Preparation of monoclonal antibody and development of an immunoassay based on immunomagnetic bead for the acenaphthene[D].Changchun:Jilin University,2016:25-28.(in Chinese)
[12]马秀丽,崔言顺,张秀美,等.鸡传染性法氏囊病间接ELISA诊断试剂盒的研制及初步应用[J].中国兽医学报,2003,23(5):424-426.MA Xiu-li,CUI Yan-shun,ZHANG Xiu-mei,et al.Development and validation of indirect en zyme-linked immunosorbent assay test kit for detection of IBDV antibody[J].Journal of Veterinary Medicine,2003,23(5):424-426.(in Chinese)
[13]郭慧芳.基于可重复使用免疫磁珠的抗体检测方法建立及可重复使用磁性微流体蛋白芯片初步设计[D].西安:第四军医大学,2006:5-9.GUO Hui-fang.A new method to detect antibodies based on immunomagnetic beads that can be used to repeatedly and the primary frame of the micro-hydromagnet proteinic-chip that can be used repeatedly[D].Xi’an:Fourth military Medical University,2006:5-9.(in Chinese)
[14]刘华洁,杜希宁,梅永杰,等.传染性法氏囊病病毒VP3基因在昆虫细胞中的表达及其ELISA抗原反应性的分析[J].中国兽医科学,2015,45(3):227-234.LIU Hua-jie,DU Xi-ning,MEI Yong-jie,et al.Expression of VP3 gene of infectious bural disease virus in insect cells and analysis of antigenicity for VP3-ELISA[J].Chinese Veterinary Science,2015,45(3):227-234.(in Chinese)
[15]刘婷.IBDV主要结构蛋白VP2、VP1原核表达及其ELISA抗体检测试剂盒的研发[D].南宁:广西大学,2016:53-58.LIU Ting.Prokaryotic expression of IBDV major structural protein VP2,VP1 and development of ELISA antibody detection kit[D].Nanning:Guangxi University,2016:53-58.(in Chinese)
[16]SHIRVANI E,PALDURAI A,MANOHARAN V K,et al.A recombinant newcastle disease virus(NDV)expressing protein of infectious bronchitis virus(IBV)protects chickens against IBV and NDV[J].Scientific Reports,2018(8):521-527.
[17]IRAM M,SHI W,WANG L,et al.Immunogenicity and protective efficacy of orally administered recombinant Lactobacillus plantarum expressing VP2 protein against IBDV in chicken[J].Journal of Applied Microbiology,2018,32(3):6-8.
[18]宋军,黄成斌,单雪芹,等.传染性法氏囊病毒VP2蛋白的表达及间接ELISA抗体检测方法的建立[J].安徽农业大学学报,2011,38(4):633-636.SONG Jun,HUANG Cheng-bin,SHAN Xue-qin,et al.Establishment of an indirect ELISA method using a infectious bursal disease virus VP2 protein as antigen[J].Journal of Anhui Agricultural University,2011,38(4):633-636.(in Chinese)
[19]王晓丽,诸玉梅,夏兴霞,等.传染性法氏囊病病毒VP2单克隆抗体夹心ELISA检测试剂盒的研制[J].中国兽医学报,2014,34(4):530-535.WANG Xiao-li,ZHU Yu-mei,XIA Xing-xia,et al.Development of sandwich ELISA Kit for detection of infectious bursal disease virus VP2 monoclonal antibody[J].Chinese Journal of Veterinary Medicine,2014,34(4):530-535.(in Chinese)
[20]SINGH N K,DEY S,MADHAN M C,et al.Evaluation of four enzyme linked immunosorbent assays for the detection of antibodies to infectious bursal disease in chickens[J].Journal of Virological Methods,2010,165(2):277-282.
[21]FAHEY K J,O'DONNELL I J,AZAD A A.Characterization by Western-blotting of the immunogens of infectious bursal disease virus[J].Journal of General Virology,1985,66(Pt7):1479-1488.
[22]王慧宁.IBDV VP2-VP5-IgY Fc融合蛋白在毕赤酵母的表达及其免疫原性的研究[D].泰安:山东农业大学,2018:2-6.WANG Hui-ning.Expression and immunogenicity of IBDV VP2-VP5-IgY Fc fusion protein in Pichia pastoris[D].Taian:Shandong Agricultural University,2018:2-6.(in Chinese)
[23]王晓丽,王永山,欧阳伟,等.传染性法氏囊病病毒VP2结构蛋白抗体ELISA检测方法的建立[J].中国兽医学报,2018,38(10):1864-1871.WANG Xiao-li,WANG Yong-shan,OUYANG Wei,et al.Establishment of an ELISA method for the detection of IBDV antibody by using the viral specific VP2 structural proteins[J].Journal of Veterinary Medicine,2018,38(10):1864-1871.(in Chinese)
[24]郑玲燕.传染性法氏囊病毒VP2基因的克隆、表达及ELISA抗体检测方法的建立[D].武汉:华中农业大学,2006:123-127.ZHENG Ling-yan.Cloning and expression of VP2gene of infectious bursal virus and preliminary development of ELISA antibody detecting[D].Wuhan:Huazhong Agricultural University,2006:123-127.(in Chinese)