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

Expression of Mycobacterium avium subsp. paratuberculosis MAP1345, MAP0862 and its primary application in indirect ELISA

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

Key words:  Mybobacterium avium subsp. paratuberculosis  Clone  Tandem expression  Indirect ELISA  Specificity  Clone  Tandem expression  Indirect ELISA  Specificity  Coincidence rate

摘要:

为了探索MAP1345与MAP0862融合表达蛋白在牛副结核病血清学诊断中的作用。使用DNASTAR软件分析MAP1345和MAP0862蛋白中具有良好抗原性的副结核特异性氨基酸序列。采用重叠PCR（splicing by overlap extension-PCR）方法将以上两段序列连接并克隆至原核表达载体pET-32a。将重组质粒转化进入BL21(DE3)感受态细胞，并对目的蛋白进行诱导表达和纯化，进一步采用Western Blot 分析目的蛋白的反应原性。将纯化后的目的蛋白定量后包被酶标板，经过一系列条件优化，初步建立了基于MAP0862-1345的间接ELISA诊断方法。使用该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、健康阴性牛血清检测，结果表明该方法具有较好的特异性；其与IDEXX副结核病抗体检测试剂盒对300份临床血清样本检测，符合率为92.7%。

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