Several tools were applied in the M.bovis protein interaction investigation. First,the yeast two-hybrid was ap-plied to verify two protein interactions in-vivo. Second,mutant strains were constructed to elucidate the mechanism of protein function. The strategies include:(1) amplification of knockout DNA;(2)preparation ofMycobacterium/pJV53 competent cells;(3)transportation of knockout DNA to competent cells;(4)screening and identification of positive colonies. The third,GST pull-down was applied to investigate known protein and target protein interaction. RD1 was in-vestigated intensively inM. bovis protein interactions. The mechanisms were found such as that dimmer was formed by ESAT-6 and CFP10,interaction was existed in Rv3873,Rv3866, Rv3868 and CFP-10,and ESAT-6 achieved its function only after it was combined with CFP-10. These findings suggest that elucidation of the interactions could facilitate the development of efficient diagnostic reagents and vaccines for bovine tuberculosis.%目前有多种技术应用于结核分枝杆菌蛋白相互作用的研究:一是酵母双杂交技术,可以在生物体内验证2个蛋白的相互作用.二是构建分枝杆菌基因突变株,研究蛋白作用的机理,其基本策略为,基因敲除双链DNA的扩增;牛结核分枝杆菌/pJV53感受态细胞的制备;将基因敲除双链DNA片段转化至牛结核分枝杆菌细胞中;筛选、鉴定阳性菌落.三是通过GST pull-down试验检测已知蛋白和靶蛋白的相互作用.目前,研究牛分枝杆菌蛋白基因相互作用较多是RD1区基因.研究表明,阐明分枝杆菌蛋白的作用机理有助于进一步研发高效的牛结核病诊断试剂和疫苗.
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