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本研究以牛分枝杆菌H37Rv基因组DNA为模板,扩增esat6和cfp10基因,构建重组表达载体p ET-32a(+)-esat6和p ET-32a(+)-cfp10,并通过原核表达系统分别表达重组esat6和cfp10蛋白,经Ni-NTA亲和层析法纯化后,对目的蛋白进行SDS-PAGE和Western blot验证,BCA法测定目的蛋白浓度,最后将east6和cfp10蛋白混合后用于牛结核病临床检测,并对检测数据进行分析。结果表明,牛分枝杆菌esat6和cfp10蛋白在大肠杆菌系统中获得高效表达;其混合蛋白应用于结核病检测具有较高的特异性,且在皮肤变态反应和IFN-γ释放试验两种检测方法中具有较高的符合率。说明相较于提纯结核菌素(PPD),esat6和cfp10混合蛋白具有较高的特异性和稳定性,将有希望替代PPD用于牛结核病检测。
In this study, esat6 and cfp10 genes were amplified by using Mycobacterium bovis H37Rv genomic DNA as a template, and the recombinant expression vectors p ET-32a (+) - esat6 and p ET-32a (+) - cfp10 were constructed. Expression of recombinant esat6 and cfp10 protein, purified by Ni-NTA affinity chromatography, the target protein was verified by SDS-PAGE and Western blot, BCA method to determine the concentration of the target protein, and finally east6 and cfp10 protein for bovine tuberculosis Clinical testing, and testing data analysis. The results showed that the mycobacterium bovis esat6 and cfp10 protein were highly expressed in E.coli. The mixed protein was used in the detection of tuberculosis with high specificity, and in skin allergy and IFN-γ release test two detection methods Has a high coincidence rate. This indicates that PPD is a promising alternative to PPD for the detection of bovine tuberculosis (TB) because of its high specificity and stability compared with purified PPD, esat6 and cfp10.