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目的克隆人组蛋白乙酰基转移酶TIP60β基因,应用酵母双杂交技术研究筛选与TIP60β发生相互作用的蛋白。方法运用RT-PCR方法从人脑组织克隆TIP60β cDNA,构建酵母双杂交BD诱饵载体pGBKT7-TIP60β,以其为诱饵从成人肝cDNA文库中筛选与之相互作用的阳性克隆,并对阳性克隆进行序列测定和相关生物学分析。结果诱饵载体pGBKT7-TIP60β经双酶切可释放出1443bp DNA片段,与理论值大小一致,测序比对分析表明序列正确。以其为诱饵经酵母双杂交筛选共获得32个克隆,进一步验证与测序分析,最终确认HDAC7A、DMD2、CREB1、BD73、PHF17、AR等9个克隆基因。结论以TIP60β为诱饵利用酵母双杂交系统获得9个基因编码的蛋白,它们很可能与TIP60β转录活性调节功能有关。
Objective To clone the human histone acetyltransferase TIP60β gene and study the screening of the protein interacting with TIP60β by yeast two-hybrid technique. Methods TIP60β cDNA was cloned from human brain tissue by RT-PCR. The yeast two-hybrid BD bait vector pGBKT7-TIP60β was constructed and used as bait to screen positive clones interacting with adult liver cDNA library. The positive clones were sequenced Determination and related biological analysis. Results The bait vector pGBKT7-TIP60β was digested by double enzyme to release 1443bp DNA fragment, which was consistent with the theoretical value. The sequencing analysis showed that the sequence was correct. Thirty-two clones were screened by yeast two-hybrid system as bait, and further validation and sequencing analysis confirmed the cloned genes of HDAC7A, DMD2, CREB1, BD73, PHF17 and AR. Conclusion TIP60β as a bait using yeast two-hybrid system to obtain nine genes encoding proteins, they are likely TIP60β transcriptional regulation of function.