基于鸡毒支原体热休克蛋白DnaK的间接ELISA方法的评价及初步应用
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摘要
为研究鸡毒支原体(MG)重组热休克蛋白DnaK (rDnaK)的免疫反应原性及其作为ELISA方法中诊断抗原的应用价值,本研究将已构建的含有MG DnaK基因的重组质粒p ET-30a-DnaK进行原核表达纯化,以多份MG的标准阳性血清和阴性血清作为一抗,进行western blot分析,结果显示r DnaK与MG的阳性血清发生特异性反应,与MG的阴性血清无反应条带,表明r DnaK具有较好的免疫反应原性。将其作为包被抗原包被酶标板,用HRP标记的兔抗鸡Ig G作为酶标二抗,分别对抗原包被浓度、血清稀释度、酶标二抗稀释度及工作时间等反应条件进行了优化,建立了一种稳定检测MG抗体的间接ELISA方法。对该方法进行评估,灵敏性试验结果显示该方法与进口试剂盒相当;特异性试验结果显示包被抗原r DnaK不与NDV、IBV、ILTV等几种常见的禽呼吸道疾病的阳性血清发生交叉反应;重复性试验结果显示,批内变异系数为1.6%～5.15%,批间变异系数为2.9%～5.98%;符合率试验结果显示该方法与进口试剂盒的总体符合率为90.4%;抗体持续期试验结果显示该方法最早能在鸡群免疫MG疫苗后一周检测到MG抗体,表明该诊断方法适用于MG感染的早期检测。采用该方法检测了来自全国5个省份的568份临床样品,其结果显示MG的感染率在23%～51%,对我国MG的流行状况做了初步的调查。
The main aim of this research was to study the immunogenicity of r Dnak and the application value as coating antigen in ELISA. The r Dnak of Mycoplasma gallisepticum(MG) was expressed in E.coli with the recombinant plasmid pET-30 a-DnaK. The product was analyzed by western blot with MG positive and negative serum and the result showed that it had a strong immunogenicity with positive serum and could not react with negative serum. Then the purified r Dnak was used as coating antigen for ELISA and HRP-tagged rabbit anti-chicken Ig G as the second antibody, the concentration of coating antigen,dilution of sera sample and second antibody were optimized as well as their incubation time. As a result, an indirect ELISA for detecting MG Ig G antibody was established. The sensitivity tests showed that the method with the same sensitivity to the import commercial detection kit; The specific assay revealed that there was no cross-reaction with the antisera against NDV, IBV, ILTV and so on; The coincidence rate test showed that the overall compliance rate of the method with the imported kit was 90.4%; The repeatability test showed that the CV of intra-and inter-assay were 1.6%-5.15% and 2.9%-5.98%, respectively; The Ig G antibody duration assay in the sera of chicken immunized with attenuated vaccine indicated that the specific Ig G antibody could be detected one week after immunization, which is important for the diagnosis of MG at early infection. With the indirect ELISA, the positive rates of 568 clinical sera samples from five provinces were between 23% and 51%, which showed a preliminary epidemiological surveys of MG infection.
The main aim of this research was to study the immunogenicity of r Dnak and the application value as coating antigen in ELISA. The r Dnak of Mycoplasma gallisepticum(MG) was expressed in E.coli with the recombinant plasmid pET-30 a-DnaK. The product was analyzed by western blot with MG positive and negative serum and the result showed that it had a strong immunogenicity with positive serum and could not react with negative serum. Then the purified r Dnak was used as coating antigen for ELISA and HRP-tagged rabbit anti-chicken Ig G as the second antibody, the concentration of coating antigen,dilution of sera sample and second antibody were optimized as well as their incubation time. As a result, an indirect ELISA for detecting MG Ig G antibody was established. The sensitivity tests showed that the method with the same sensitivity to the import commercial detection kit; The specific assay revealed that there was no cross-reaction with the antisera against NDV, IBV, ILTV and so on; The coincidence rate test showed that the overall compliance rate of the method with the imported kit was 90.4%; The repeatability test showed that the CV of intra-and inter-assay were 1.6%-5.15% and 2.9%-5.98%, respectively; The Ig G antibody duration assay in the sera of chicken immunized with attenuated vaccine indicated that the specific Ig G antibody could be detected one week after immunization, which is important for the diagnosis of MG at early infection. With the indirect ELISA, the positive rates of 568 clinical sera samples from five provinces were between 23% and 51%, which showed a preliminary epidemiological surveys of MG infection.
引文
[1]Abdelmoumen M B,Bejaoui A A,Oussaeif L,et al.A recombinant antigen-based ELISA for the simultaneous differential serodiagnosis of Mycoplasma gallisepticum,Mycoplasma synoviae,and Mycoplasma meleagridis infections[J].Avian Dis,2008,52(2):214-221.
[2]刘月凤.鸡毒支原体诊断研究新进展[J].陕西农业科学,2011(01):93-95.
[3]孟冬霞,贺东昌,武果桃,等.山西省鸡毒支原体感染血清学调查[J].中国兽医杂志,2011,(08):45-47.
[4]孔意端,陈峰,壞理克,等.广东地区鸡毒支原体血清学调查[J].广东畜牧兽医科技,2010,(01):21-23.
[5]Davis K L,Wise K S.Site-specific proteolysis of the MALP-404 lipoprotein determines the release of a soluble selective lipoprotein-associated motif-containing fragment and alteration of the surface phenotype of Mycoplasma fermentans[J].Infect Immun,2002,70(3):1129-1135.
[6]Minion F C,Lefkowitz E J,Madsen M L,et al.The genome sequence of Mycoplasma hyopneumoniae strain 232,the agent of swine mycoplasmosis[J].J Bacteriol,2004,186(21):7123-7133.
[7]Jun A,Yuji H,Motomi I,et al.Expression of exogenous genes under the control of endogenous HSP70 and CAB promoters in the closterium peracerosum strigosum littorale complex[J].Plant Cell Physiol,2008,49(4):625-632.
[8]李媛,张建华,等.猪肺炎支原体中国分离株热休克蛋白Hsp70(Dnak)的全基因克隆及C末端基因的表达与免疫活性分析[J].中国预防兽医学报,2008,30(3):169-173.
[9]Feberwee A,Wit J J,Landman W J.Induction of eggshell apex abnormalities by Mycoplasma synoviae:field and experimental studies[J].Avian Pathol,2009,38(3):77-85.
[10]包世俊.滑液支原体MVU1853株免疫相关膜蛋白的初步分析[J].畜牧兽医学报,2017,48(2):316-323.
[11]Buyuktanir O,Yildirim T,Yakicier C,et al.A recombinant PvpA protein-based diagnostic prototype for rapid screening of chicken Mycoplasma gallisepticum infections[J].Vet Microbiol2008,129:139-149.
[12]Ozlem B,Oktay G,Nevzat Y,et al.Bi-antigenic immunoassay models based on the recombinant Pvp A proteins for Mycoplasma gallisepticum diagnosis in chickens[J].Vet Diagn Invest,2010,22(3):908-913.
[13]Abdelmoumen M B,Bejaoui A A,Oussaeif L,et al.A recombinant antigen-based ELISA for the simultaneous differential serodiagnosis of Mycoplasma gallisepticum,Mycoplasma synoviae,and Mycoplasma meleagridis infections[J].Avian Dis,2008,52:214-221.
[14]周云雷,李健.以重组PvpA蛋白作为抗原的鸡毒支原体抗体间接ELISA检测方法的建立[J].中国预防兽医学报,2011,33(9):713-717.
[2]刘月凤.鸡毒支原体诊断研究新进展[J].陕西农业科学,2011(01):93-95.
[3]孟冬霞,贺东昌,武果桃,等.山西省鸡毒支原体感染血清学调查[J].中国兽医杂志,2011,(08):45-47.
[4]孔意端,陈峰,壞理克,等.广东地区鸡毒支原体血清学调查[J].广东畜牧兽医科技,2010,(01):21-23.
[5]Davis K L,Wise K S.Site-specific proteolysis of the MALP-404 lipoprotein determines the release of a soluble selective lipoprotein-associated motif-containing fragment and alteration of the surface phenotype of Mycoplasma fermentans[J].Infect Immun,2002,70(3):1129-1135.
[6]Minion F C,Lefkowitz E J,Madsen M L,et al.The genome sequence of Mycoplasma hyopneumoniae strain 232,the agent of swine mycoplasmosis[J].J Bacteriol,2004,186(21):7123-7133.
[7]Jun A,Yuji H,Motomi I,et al.Expression of exogenous genes under the control of endogenous HSP70 and CAB promoters in the closterium peracerosum strigosum littorale complex[J].Plant Cell Physiol,2008,49(4):625-632.
[8]李媛,张建华,等.猪肺炎支原体中国分离株热休克蛋白Hsp70(Dnak)的全基因克隆及C末端基因的表达与免疫活性分析[J].中国预防兽医学报,2008,30(3):169-173.
[9]Feberwee A,Wit J J,Landman W J.Induction of eggshell apex abnormalities by Mycoplasma synoviae:field and experimental studies[J].Avian Pathol,2009,38(3):77-85.
[10]包世俊.滑液支原体MVU1853株免疫相关膜蛋白的初步分析[J].畜牧兽医学报,2017,48(2):316-323.
[11]Buyuktanir O,Yildirim T,Yakicier C,et al.A recombinant PvpA protein-based diagnostic prototype for rapid screening of chicken Mycoplasma gallisepticum infections[J].Vet Microbiol2008,129:139-149.
[12]Ozlem B,Oktay G,Nevzat Y,et al.Bi-antigenic immunoassay models based on the recombinant Pvp A proteins for Mycoplasma gallisepticum diagnosis in chickens[J].Vet Diagn Invest,2010,22(3):908-913.
[13]Abdelmoumen M B,Bejaoui A A,Oussaeif L,et al.A recombinant antigen-based ELISA for the simultaneous differential serodiagnosis of Mycoplasma gallisepticum,Mycoplasma synoviae,and Mycoplasma meleagridis infections[J].Avian Dis,2008,52:214-221.
[14]周云雷,李健.以重组PvpA蛋白作为抗原的鸡毒支原体抗体间接ELISA检测方法的建立[J].中国预防兽医学报,2011,33(9):713-717.