基于牛支原体重组蛋白rMbovP730的间接ELISA方法的建立与应用  

Establishment and application of an indirect ELISA method based on recombinant protein rMbovP730 of Mycoplasma bovis

作　　者：李祥慧 徐明国 马海龙 杨中莲 王勇[1,2,3] 马忠臣 陈创夫 LI Xiang-hui;XU Ming-guo;MA Hai-long;YANG Zhong-lian;WANG Yong;MA Zhong-chen;CHEN Chuang-fu(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;International Joint Research Center for Animal Health,Shihezi 832003,China;Key Laboratory of Animal Disease Prevention and Control Corps,Shihezi 832003,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]动物健康养殖国际联合研究中心,新疆石河子832003 [3]动物疾病防控兵团重点实验室,新疆石河子832003

出　　处：《中国预防兽医学报》2024年第12期1255-1260,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：兵团重大科技项目(2017AA003)。

摘　　要：为获得具有良好反应原性和免疫原性的牛支原体重组蛋白rMbovP730,并建立基于rMbovP730的牛支原体抗体的间接ELISA检测方法,本研究经PCR扩增牛支原体MbovP730基因后,利用原核表达方式获得重组蛋白rMbovP730,利用牛支原体阳性血清经western blot鉴定其反应原性。以该重组蛋白为包被抗原,采用棋盘法对各反应条件优化后建立牛支原体抗体的间接ELISA检测方法。结果显示,原核表达的重组蛋白rMbovP730约35 ku,可以与牛支原体阳性血清发生特异性反应;间接ELISA检测方法优化结果显示,重组rMbovP730蛋白最佳包被浓度为4μg/mL,待检血清最佳稀释度为1:100,兔抗牛IgG-HRP的最佳稀释度为1:10000。利用建立的间接ELISA方法检测牛支原体、牛布鲁氏菌、牛病毒性腹泻病毒、牛轮状病毒的阳性血清,结果显示,除牛支原体阳性血清检测结果为阳性外,其余病原体阳性血清均为阴性,该方法特异性较强;将牛支原体的阳性血清2倍倍比稀释(1:20~1:2560)后利用该方法检测,结果显示,该阳性血清以1:2560稀释后检测结果仍为阳性,敏感性较高;利用同一批次和不同批次制备的重组蛋白包被后进行批内、批间重复性试验,结果显示,批内、批间重复性试验的变异系数均小于10%,重复性较好。利用建立的间接ELISA方法对130份临床牛血清样品检测,结果显示检出阳性血清58份,阴性血清72份,阳性率为44.6%;商品化ELISA试剂盒检出阳性血清28份,阴性血清102份,阳性率为21.5%;二者的总符合率为76.9%。本研究首次基于rMbovP730建立的间接ELISA方法特异性强、敏感性高、重复性好,为研制牛支原体抗体检测试剂盒奠定了基础。In order to obtain the Mycoplasma bovis recombinant protein rMbovP730 with good reactogenicity and immunogenicity and to establish an indirect ELISA detection method for Mycoplasma bovis antibodies based on the rMbovP730,this study carried out molecular cloning of the Mycoplasma bovis MbovP730 gene and obtained the recombinant protein rMbovP730 using prokaryotic expression.An indirect ELISA detection method for Mycoplasma bovis antibodies was established using this protein as the coating antigenby optimizing various reaction conditions.The results showed that the prokaryotic expressed rMbovP730 recombinant protein is about 35ku and can specifically react with Mycoplasma bovis-positive serum.In the indirect ELISA method,the optimal coating concentration of recombinant protein rMbovP730 is 4μg/mL,the optimal dilution of the serum to be tested is 1100,and the optimal dilution of rabbit anti-bovine HRP-IgG is 110000.The specificity,sensitivity,and reproducibility of the method were evaluated and applied to clinical sample detection.Specificity test of the indirect ELISA method showed that the positive sera for Mycoplasma bovis were positive,while the positive sera for Brucella bovis,bovine viral diarrhea virus,and bovine rotavirus were all negative,indicating the good specificity of this method.The sensitivity test showed that the positive sera of Mycoplasma bovis is still positive after being diluted at 12560,demonstrating its high sensitivity.The intra-batch and inter-batch tests showed that the coefficient of variation is less than 10%,indicating good repeatability.Indirect ELISA was used to detect 130 clinical bovine serasamples,and 58 positive sera and 72 negative sera were detected,with a positive rate of 44.6%.In contrast,a commercial ELISA kit detected 28 positive sera and 102 negative sera,with a positive rate of 21.5%.The coincidence rate between the two methods is 76.9%.This study established an indirect ELISA method based on the recombinant protein rMbovP730,which has high sensitivity and good reproducib

关 键 词：牛支原体 重组蛋白rMbovP730 原核表达 ELISA 

分 类 号：S852.6[农业科学—基础兽医学]