口蹄疫病毒非结构蛋白抗体时间分辨荧光免疫分析检测方法的建立
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摘要
为建立快速定量检测羊血清中口蹄疫病毒非结构蛋白抗体的方法,本研究通过优化抗原表达条件等步骤,在大肠杆菌原核表达系统中获得了可溶性的3A-3B1-3B2融合蛋白,基于纯化的可溶性融合蛋白建立了口蹄疫病毒非结构蛋白抗体时间分辨荧光免疫分析检测试剂盒。该方法能够检测羊血清中的口蹄疫病毒非结构蛋白抗体,敏感性高,特异性强,对其他相关的羊类病原无交叉反应,其组内与组间变异系数分别低于10%和15%,具有良好的重复性。对300份临床血清样品进行检测,同Procheck公司的口蹄疫非结构蛋白抗体试剂盒进行比较,阳性样品符合率为98%,阴性样品符合率为92%,总符合率为95%。重复性试验组内与组间变异系数均小于10%。口蹄疫病毒非结构蛋白抗体时间分辨荧光免疫分析检测方法同传统的ELISA方法相比,特异性相当、敏感性更高,操作简单、快速,具有较高的应用推广价值。
To establish the time-resolved fluoroimmunoassay method for the detection of foot-andmouth disease virus antibodies in goat serum,this research obtained the soluble 3 A-3 B1-3 B2 fusion protein in Escherichia coli,through exploration of protein expression conditions.Further based on the purified fusion protein,a time-resolved fluoroimmunoassay detection method for foot-and-mouth disease virus antibodies was established.This method could detect the antibodies against foot-and-mouth disease virus in goat serum quickly and quantitatively.It had high sensitivity and specificity,and had no cross reaction to other related goat pathogens.The coefficient of variation intra batch and inter batch were all below 10%.A total of 300 clinical serum samples were tested,and the ELISA kit(Procheck)was used for comparison.The coincidence rate of the positive samples was 98%,the coincidence rate of the negative samples was 92%,and the total coincidence rate was 95%.In conclusion this method has a wide range of detection with high specificity,and has a high practical value and popularization value,compared with the traditional ELISA.
To establish the time-resolved fluoroimmunoassay method for the detection of foot-andmouth disease virus antibodies in goat serum,this research obtained the soluble 3 A-3 B1-3 B2 fusion protein in Escherichia coli,through exploration of protein expression conditions.Further based on the purified fusion protein,a time-resolved fluoroimmunoassay detection method for foot-and-mouth disease virus antibodies was established.This method could detect the antibodies against foot-and-mouth disease virus in goat serum quickly and quantitatively.It had high sensitivity and specificity,and had no cross reaction to other related goat pathogens.The coefficient of variation intra batch and inter batch were all below 10%.A total of 300 clinical serum samples were tested,and the ELISA kit(Procheck)was used for comparison.The coincidence rate of the positive samples was 98%,the coincidence rate of the negative samples was 92%,and the total coincidence rate was 95%.In conclusion this method has a wide range of detection with high specificity,and has a high practical value and popularization value,compared with the traditional ELISA.
引文
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[8]姚怀兵,赵毅,刘梦丽,等.口蹄疫病毒3C蛋白酶结构与功能及应用研究进展[J].动物医学进展,2017,38(3):102-106.YAO Huai-bing,ZHAO Yi,LIU Meng-li,et al.Advance in structure,function and application of 3C protease of foot-and-mouth disease virus[J].Progress in Veterinary Medicine,2017,38(3):102-106.(in Chinese)
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[10]HAN S C,GUO H C,SUN S Q.Three-dimensional structure of foot-and-mouth disease virus and its biological functions[J].Archives of Virology,2015,160(1):1-16.
[11]CLAVIJO A,HOLE K,LI M,et al.Simultaneous detection of antibodies to foot-and-mouth disease nonstructural proteins 3ABC,3D,3A and 3B by multiplexed Luminex assay to differentiate infected from vaccinated cattle[J].Vaccine,2006,24(10):1693-1704.
[12]孙燕燕,马米玲,周广青,等.A型口蹄疫病毒抗体金标检测试纸条的研制[J].中国兽医科学,2016,46(9):1123-1127.SUN Yan-yan,MA Mi-ling,ZHOU Guang-qing,et al.Development of gold-labeled immunochromato graphic test strip for detecting antibodies against serotype A foot-and-mouth disease virus[J].Chinese Veterinary Science,2016,46(9):1123-1127.(in Chinese)
[13]ALEXANDERSEN S,ZHANG Z,DONALDSON A I,et al.The pathogenesis and diagnosis of foot-and-mouth disease[J].Journal of Cmparative Pathology,2003,129(1):1-36.
[14]李顺琴,杨君敬,何诚,等.口蹄疫病毒细胞培养技术研究进展[J].动物医学进展,2018,39(1):90-94.LI Shun-qin,YANG Jun-jing,HE Chen,et al.Advance in cell culture techniques of of foot-and-mouth disease virus[J].Progress in Veterinary Medicine,2018,39(1):90-94.(in Chinese)
[15]LI S,GAO M,ZHANG R,et al.A mutant of infectious Asia 1 serotypefoot-and-mouth disease virus with the deletion of 10-amino-acidin the 3A protein[J].Virus Genes,2010,41(3):406-413.
[16]FOWLER V L,BASHIRUDDIN J B,MAREE F F,et al.Footand-mouth disease marker vaccine:cattle protection with a partial VP1 GeH loop deleted virus antigen[J].Vaccine,2011,29(46):8405-8411.
[2]NISHI T,ULZIIBAT G,KHANUI B,et al.Genome sequence of foot-and-mouth disease virus of serotype O lineage Ind-2001 isolated from cattle in Mongolia in2015[J].Genome Announcement,2017,5(45):1244.
[3]EHIZIBOLO D O,HAEGEMAN A,VLEESCHAUWER A R,et al.Detection and molecular characterization of foot and mouth disease viruses from outbreaks in some states of northern nigeria 2013-2015[J].Transboundary and Emerging Diseases,2017,64(6):1979-1990.
[4]MAHAPATRA M,UPADHYAYA S,AVISO S,et al.Selection of vaccine strains for serotype O foot-and-mouth disease viruses(2007-2012)circulating in Southeast Asia,East Asia and far east[J].Vaccine,2017,35(51):7147-7153.
[5]LU Z,ZHANG X,FU Y,et al.Expression of the major epitope regions of 2C integrated with the 3AB nonstructural protein of foot-and-mouth disease virus and its potential for differentiating infected from vaccinated animals[J].Journal of Virological Methods,2010,170(1-2):128-133.
[6]ELNEKAVE E,SHILO H,GELMAN B,et al.The longevity of anti NSP antibodies and the sensitivity of a3ABC ELISA-A 3 years follow up of repeatedly vaccinated dairy cattle infected by foot and mouth disease virus[J].Veterinary Microbiology,2015,178(1):14-18.
[7]ERGMANN I E,MALIRAT V,NEITZERT E,et al.Improvement of aserodiagnostic strategy for foot-and-mouth disease virus surveil-lance in cattle under systematic vaccination:a combined system of an indirect ELISA-3ABC with an enzyme-linked immunoelec-trotransfer blot assay[J].Archives of Virology,2000,145(3):473-489.
[8]姚怀兵,赵毅,刘梦丽,等.口蹄疫病毒3C蛋白酶结构与功能及应用研究进展[J].动物医学进展,2017,38(3):102-106.YAO Huai-bing,ZHAO Yi,LIU Meng-li,et al.Advance in structure,function and application of 3C protease of foot-and-mouth disease virus[J].Progress in Veterinary Medicine,2017,38(3):102-106.(in Chinese)
[9]曲哲会,王君伟.口蹄疫病毒非结构蛋白3ABC基因的原核表达及其产物的活性检测[J].中国兽医科学,2006,36(9):696-700.QU Zhe-hui,WANG Jun-wei.Prokaryotic expression of FMDV non-structural protein 3ABC gene and activities of the expressed product[J].Chinese Veterinary Science,2006,36(9):696-700.(in Chinese)
[10]HAN S C,GUO H C,SUN S Q.Three-dimensional structure of foot-and-mouth disease virus and its biological functions[J].Archives of Virology,2015,160(1):1-16.
[11]CLAVIJO A,HOLE K,LI M,et al.Simultaneous detection of antibodies to foot-and-mouth disease nonstructural proteins 3ABC,3D,3A and 3B by multiplexed Luminex assay to differentiate infected from vaccinated cattle[J].Vaccine,2006,24(10):1693-1704.
[12]孙燕燕,马米玲,周广青,等.A型口蹄疫病毒抗体金标检测试纸条的研制[J].中国兽医科学,2016,46(9):1123-1127.SUN Yan-yan,MA Mi-ling,ZHOU Guang-qing,et al.Development of gold-labeled immunochromato graphic test strip for detecting antibodies against serotype A foot-and-mouth disease virus[J].Chinese Veterinary Science,2016,46(9):1123-1127.(in Chinese)
[13]ALEXANDERSEN S,ZHANG Z,DONALDSON A I,et al.The pathogenesis and diagnosis of foot-and-mouth disease[J].Journal of Cmparative Pathology,2003,129(1):1-36.
[14]李顺琴,杨君敬,何诚,等.口蹄疫病毒细胞培养技术研究进展[J].动物医学进展,2018,39(1):90-94.LI Shun-qin,YANG Jun-jing,HE Chen,et al.Advance in cell culture techniques of of foot-and-mouth disease virus[J].Progress in Veterinary Medicine,2018,39(1):90-94.(in Chinese)
[15]LI S,GAO M,ZHANG R,et al.A mutant of infectious Asia 1 serotypefoot-and-mouth disease virus with the deletion of 10-amino-acidin the 3A protein[J].Virus Genes,2010,41(3):406-413.
[16]FOWLER V L,BASHIRUDDIN J B,MAREE F F,et al.Footand-mouth disease marker vaccine:cattle protection with a partial VP1 GeH loop deleted virus antigen[J].Vaccine,2011,29(46):8405-8411.