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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 interacting with FAM172A protein in human fetal brain cDNA library by yeast two-hybrid technique (protein-protein interaction), and lay a foundation for further study on the biological function of newly discovered protein FAM172A and its role in the disease. Methods The bait plasmid pGB-FAM172A was constructed and transformed into yeast strain Y190. Human fetal brain cDNA was transformed into bait yeast and grown in auxotrophy medium (SD / -Leu / -Trp / -His). 35 clones were selected for β-galactosidase cloning and transfer filter test. The blue clones were subjected to plasmid extraction and transformed into E. coli DH / OB for resistance screening. The plasmids were extracted from the clones and the pGB-FAM172A co-transformed yeast cells Y190 were identified and identified. The plasmid DNA was extracted and sequenced And BLAST analysis. RESULTS: The pGB-FAM172A plasmid was successfully constructed and 10 positive clones were screened. Sequencing and sequence alignment were performed respectively. Six proteins that interacted with FAM172A were successfully screened: RTCD1, MOCS2, A2M, KCNIP1, BTBD2 and TOX2 . CONCLUSION: Six proteins that may interact with FAM172A were screened by yeast two-hybrid system, providing clues for further study on the biological function of FAM172A protein.