应用酵母双杂交筛选与FAM172A相互作用蛋白的研究

Screening Proteins Interacting with FAM172A Protein by the Yeast Two Hybridization

目的应用酵母双杂交技术(蛋白与蛋白的相互作用),筛选人胎脑cDNA文库中与FAM172A蛋白相互作用的蛋白,为深入研究新发现的蛋白质FAM172A生物学功能及在疾病中的作用奠定基础。方法构建pGB-FAM172A诱饵质粒,转化酵母菌株Y190。人胎脑cDNA转化诱饵酵母菌,于营养缺陷型培养基(SD/-Leu/-Trp/-His)上生长,从中筛选到35个单克隆进行β-半乳糖苷酶克隆转移滤纸实验,对蓝色克隆者进行质粒抽提,转入大肠杆菌DH/OB,进行抗性筛选,从中提取质粒,一对一与诱饵质粒pGB-FAM172A共转酵母细胞Y190,进行验证鉴定,提取质粒DNA进行测序并进行BLAST比对分析。结果成功构建pGB-FAM172A质粒,经严格筛选,共有10个阳性克隆,分别进行测序、序列比对,成功筛选出6个与FAM172A存在相互作用的蛋白:RTCD1、MOCS2、A2M、KCNIP1、BTBD2和TOX2。结论利用酵母双杂交系统筛选出6个可能与FAM172A相互作用的蛋白,为进一步研究FAM172A蛋白的生物学功能提供了线索。

Objective To screen the proteins in human fetal brain cDNA library interacting with FAM172A protein by yeast two-hybrid system （protein-protein interactions technique）,and to lay the foundation for the further study about the biological function of the FAM172A protein and its roles in process of diseases.Methods The PGB-FAM172A bait plasmids were constructed and transformed into the yeast strain Y190.The bait yeast strains were incubated in the auxotrophic medium （SD/-Leu/-Trp/-His） after being transformed with Human fetal brain cDNA.Thirty-five monoclones were selected for the β-galactosidase colony-lift filter assay,from which blue clones were employed for plasmid extraction.The plasmids were then transformed into E.coli DH/OB.After antibiotic screening,both the extracted plasmid and the PGB-FAM172A bait plasmid were co-transformed into yeast cells Y190.At last we identified them,and extracted the DNA of the plasmid for sequencing and Blast comparative analysis.Results Ten positive clones selected strictly from the successful-constructed PGB-FAM172A plasmid were sequenced and compared.Six proteins interacted with FAM172A protein were left,including RTCD1,MOCS2,A2M,KCNIP1,BTBD2 and TOX2.Conclusion Six proteins involved in the interaction with FAM172A were selected by the yeast two-hybrid system.These findings brought some new clues for the further exploration of the FAM172A protein.