CAJ | 학술논문

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.
목전유다충기술응용우결핵분지간균단백상호작용적연구:일시효모쌍잡교기술,가이재생물체내험증2개단백적상호작용.이시구건분지간균기인돌변주,연구단백작용적궤리,기기본책략위,기인고제쌍련DNA적확증;우결핵분지간균/pJV53감수태세포적제비;장기인고제쌍련DNA편단전화지우결핵분지간균세포중;사선、감정양성균락.삼시통과GST pull-down시험검측이지단백화파단백적상호작용.목전,연구우분지간균단백기인상호작용교다시RD1구기인.연구표명,천명분지간균단백적작용궤리유조우진일보연발고효적우결핵병진단시제화역묘.
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.