应用酵母双杂交筛选与FAM172A相互作用蛋白的研究 |
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引用本文: | 张蓉,李梅芳,李连喜,俞立波,赵催春,屠印芳,陆俊茜,包玉倩,贾伟平.应用酵母双杂交筛选与FAM172A相互作用蛋白的研究[J].医学研究杂志,2014,43(4):37-41. |
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作者姓名: | 张蓉 李梅芳 李连喜 俞立波 赵催春 屠印芳 陆俊茜 包玉倩 贾伟平 |
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作者单位: | [1]苏州大学医学部,215123 [2]上海交通大学附属第六人民医院内分泌代谢科、上海市糖尿病研究所、上海市糖尿病重点实验室、上海市糖尿病临床医学中心、上海市代谢病临床医学中心,200233 |
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基金项目: | 国家自然科学基金面上项目(81170759) |
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摘 要: | 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.
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关 键 词: | FAM172A基因 α2巨球蛋白 酵母双杂交系统 蛋白相互作用 |
Screening Proteins Interacting with FAM172A Protein by the Yeast Two Hybridization |
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Institution: | Zhang Rong,Li Meifang Li Lianxi( 1.Soochow University, Medical College, Jiangsu 215123, China;) |
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Abstract: | 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. |
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Keywords: | FAM172A gene Alpha2-macroglobulin Yeast two-hybrid system Protein-protein interaction |
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