利用酵母双杂交系统筛选TRADD的互作牛分枝杆菌蛋白

为研究牛分枝杆菌抑制肿瘤坏死因子介导的细胞凋亡来逃避宿主免疫反应的机制，本试验采用酵母双杂交系统在牛分枝杆菌中筛选可与肿瘤坏死因子受体1相关死亡域蛋白（TRADD）相互作用的蛋白。通过限制性内切酶Sau3AⅠ部分消化牛分枝杆菌基因组，回收片段随机插入pGADT7载体中，转化大肠杆菌DH5α感受态细胞，构建牛分枝杆菌基因组文库。PCR扩增人tradd基因，将扩增产物克隆于pGBKT7载体上，构建重组诱饵质粒pGBKT7-tradd，转化酵母菌Y2HGold。用诱饵质粒pGBKT7-tradd对牛分枝杆菌基因组文库进行筛选，以获得与TRADD互作的阳性候选克隆；提取阳性候选克隆中的质粒，经测序和同源性比对分析，获得与TRADD互作的牛分枝杆菌蛋白的生物学信息。结果显示，构建的文库滴度为2×10⁶ CFU，平均插入片段在1.5 kb左右，文库重组率>95%。经Western blotting验证，诱饵质粒pGBKT7-tradd可在酵母菌中表达诱饵蛋白TRADD，且TRADD的表达对酵母菌无毒性，不会在酵母菌中自激活。应用酵母双杂交系统初步筛选出20个与TRADD互作的阳性克隆，经复筛、测序和BLAST对比，最终发现7个基因序列。本研究应用酵母双杂交技术成功筛选到7个与TRADD互作的牛分枝杆菌蛋白，为进一步研究牛分枝杆菌对细胞凋亡的抑制机制提供线索。

Screening of Mycobacterium bovis Proteins Interacting with TRADD by Yeast Two-hybrid System

To investigate the mechanism of Mycobacterium bovis inhibiting apoptosis,proteins interacted with human TNFR1 associated death domain (TRADD) were screened by yeast two-hybrid system.In order to construct the genome library,the genome of Mycobacterium bovis was digested by Sau3AⅠ,and then inserted into the vector pGADT7,and transformed into E.coli DH5α.tradd gene was amplified by PCR and inserted into the vector pGBKT7.The recombinant vector named pGBKT7-tradd was transformed into yeast Y2HGold.The genome library of Mycobacterium bovis was screened to obtain positive clones interacting with human TRADD.The positive clones were confirmed by DNA sequencing and BLAST.The results showed that the titer of library was 2×10⁶ CFU with the average of inserted fragments about 1.5 kb,and the recombinant rate was >95%.TRADD expression without toxicity and autoactivation in yeast was detected by Western blotting.Seven proteins interacting with TRADD were obtained by homology analysis from 20 positive colonies.The candidate proteins in the study were provided the clue to research the infection mechanism of Mycobacterium bovis.