【摘要】 目的筛选细胞极化蛋白PALS1的结合蛋白,进一步探讨PALS1的功能及其分子机制。方法将PALS1的全长cDNA序列克隆到载体pGBKT7中,重组的pGBKT7-PALS1质粒转入酵母AH109细胞,细胞置于SD/-Trp培养基上生长,阳性细胞再用Hela细胞的cDNA文库质粒转化,生长于SD/-Trp/-Leu/-His/X-α-gal,显蓝色的克隆为阳性。阳性克隆细胞中的DNA被抽提后转化细菌细胞,然后抽提DNA进行酶切分型。合适的类型测序后进行BLAST检索分析。结果筛选到了13个阳性克隆,其中有2个是已知蛋白的基因即ATRAP和GLT28D1。结论利用酵母双杂交系统成功克隆了PALS1的结合蛋白的基因,为进一步研究PALS1蛋白的功能提供了有益的启示。更多还原

【Abstract】 Objective To screen proteins interacting with PALS1 by yeast two-hybrid technique, and to explore the functions and molecular mechanisms of PALS1.Methods The full-length DNA fragment of PALS1 was inserted into the “bait” vector pGBKT7 in frame.The recombinant pGBKT7-PALS1 was then transformed into yeast strain AH109 and cultured in SD/-Trp medium, followed by transformation of human Hela cell cDNA library. Candidate positive clones were assayed by α-galactosidase activity on SD/-Trp/-Leu/-His. Confirmed positive clone plasmids were sequenced and retrieved with Blast Program. Results 13 positive clones were obtained by yeast two-hybrid system, and two genes of them were known genes which encode ATRAP or GLT28D1. Conclusion Our result shows that ATRAP or GLT28D1 might interact with PALS1 in yeast cells, and suggest the PALS1 may have broader functions than recognized.更多还原