酵母双杂交系统筛选与RapGAP相互作用的蛋白质

Screening for Rap GAP interacting proteins by Yeast two-hybrid system

目的筛选与Rap GAP相互作用的蛋白质,为进一步研究人源Rap1GAP介导的信号转导通路、揭示其与肿瘤的关系提供实验依据。方法选用与Rap1GAP同源的来自美丽线虫的Rap GAP作为饵蛋白,以来源于美丽线虫的c DNA文库作为靶蛋白,应用p PC97、p PC86组成的酵母双杂交系统筛选c DNA文库中与Rap GAP相互作用的蛋白质。结果通过营养缺陷平板（-LTH）筛选出63个拟似阳性菌落。经过Lac Z鉴定,19个菌落为阳性,其中7个为强阳性。提取来自19个酵母菌落中的重组DNA,经PCR扩增,12个菌落出现阳性结果。将该19个重组DNA分别电转化入DH5α细菌,涂板培养后,每板挑取4~10个克隆,通过Sal I和Not I双酶切鉴定进行阳性克隆筛选。将阳性克隆的重组DNA进行序列测定。测序结果与Gen Bank比较,其中4个克隆的DNA片段为Y39b6a基因片段、2个为Rap GAP、1个为苯丙氨酸-4-羟化酶、1个为细胞色素C氧化酶,还有1个DNA片段编码美丽线虫特有的小分子蛋白的基因片段,其余11个DNA片段不编码已知蛋白质。结论初步筛选出与Rap GAP相互作用的蛋白质,特别是其中有2个克隆为Rap GAP,提示Rap GAP可能以二聚体的方式存在。

Objective To screen potential proteins interacting with Rap GAP,in order to investigate the Rap1 GAPmediated signal pathways and discover the relationship between Rap1 GAP and tumorigenesis. Methods Yeast two hybrid（ Y2H） system was employed to look for the potential proteins interacting with Rap GAP,a homological protein of human Rap1 GAP from C. elegans. In Y2 H system,Rap GAP from C. elegans was used as a bait and c DNA library from C. elegans was as a target. Results 63 potential positive colonies were obtained by-LTH screening.Among them,19 colonies were positive after Lac Z screening. The recombinant DNAs were isolated from yeast cells of the 19 colonies separately,and were checked by PCR; 12 out of the 19 shown a positive result. The recombinant DNAs were electro-transformed into DH5（ cells. After screening with LB plates containing antibody,4 to 10 colonies from each plate were screened for positive clones by Sal I and Not I digestion. Then the recombinant DNAs from positive clones were sequenced. By compared with Gen Bank,four DNA fragments were Y39b6 a,two were Rap GAP,one was phenylalanine-4-hydroxylase,one was cytochrome C oxidase,one was a gene fragment encoding a small-molecule protein which specially belongs to C. elegans,and the remaining 11 were DNA fragments which do not encode any known proteins. Conclusion The proteins interacting with Rap GAP were screened,among thixh two molecules were Rap GAPs,suggestting that Rap GAP from C. elegans may exists in a form of dimer in cells.