| 副结核分枝杆菌MAP0862、MAP1345基因的串联表达及其在间接ELISA中的初步应用 |
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| 引用本文： | 张 振,丁家波.副结核分枝杆菌MAP0862、MAP1345基因的串联表达及其在间接ELISA中的初步应用[J].中国兽药杂志,2016,50(6):16-21. |
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| 作者姓名： | 张 振 丁家波 |
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| 作者单位： | 山东农业大学；中国兽医药品监察所,中国兽医药品监察所 |
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| 基金项目： | 北京市科技新星计划(xx2013099)；现代农业人才支撑计划项目 |
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| 摘 要： | 为探索副结核特异性蛋白MAP0862与MAP1345在牛副结核病血清学诊断中的作用,将通过串联表达获得的融合蛋白MAP0862-1345纯化定量后包被酶标板,经过对反应条件的优化,初步建立了基于融合蛋白MAP0862-1345的间接ELISA诊断方法。使用建立的ELISA方法对牛副结核病阳性血清、牛结核病阳性血清、牛布病阳性血清、卡介苗免疫牛血清、健康牛血清检测的结果表明其具有较好的特异性;使用该方法与IDEXX副结核病抗体检测试剂盒共同对300份临床血清样本检测,总符合率为92.7%。
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| 关 键 词： | 副结核分枝杆菌 克隆 串联表达 间接ELISA 特异性 符合率 |
| 收稿时间： | 2016/4/26 0:00:00 |
| 修稿时间： | 6/2/2016 12:00:00 AM |
Expression of Mycobacterium avium subsp. paratuberculosis MAP1345, MAP0862 and its primary application in indirect ELISA |
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| Institution: | Shandong Agriculture University , China Institute of Veterinary Drug Control, |
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| Abstract: | To explore the effect of fusion protein of MAP1345 and MAP0862 in the diagnosis of Bovine paratuberculosis, the specific sequence of MAP1345 and MAP0862 with good antigenicity was anlysised by DNASTAR. The fusion gene was acquired by overlap extension PCR and inserted into pET-32a expression vector, Then the recombinant plasmid was transformed into Escherichia coli BL21(DE3).S The fusion protein was expressed and analyzed by SDS-PAGE. The antigenicity of fusion protein was also analyzed by Western blot after purification. An indiret ELISA method was primary established after a series of optimization by using the purified protein as coating antigen. The method was proved to be specific by verification with MAP antiserum, Brucella antiserum, Bovine tuberculosis (TB) antiserum, BCG antiserum, and negative serum. The coincidence rate of MAP0862 indiret ELISA with commercial kit was 92.7% by detection of 300 serum samples with both methods |
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| Keywords: | Mybobacterium avium subsp paratuberculosis Clone Tandem expression Indirect ELISA Specificity Clone Tandem expression Indirect ELISA Specificity Coincidence rate |
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