核基质MINT N端片段(365～1084aa)相互作用蛋白的筛选及鉴定

Screening and identification of nuclear matrix protein MINT N-terminal fragment (365-1084aa) interacting proteins

目的 :确定核蛋白MINT(Msx2 interactingnucleartargetProtein)的功能及作用机制 .方法 :利用酵母双杂交的方法 ,以MINTN端第 36 5～ 1 0 84位氨基酸 (MINT F2 )为诱饵用酵母双杂交法筛选小鼠胚胎 9日的cDNA文库 .用诱饵质粒和文库先后转化酵母宿主后 ,对 1 .0× 1 0 8个酵母克隆进行营养筛选和 β 半乳糖苷酶筛选 ,获得 1 2 8个阳性克隆 .并提取质粒进行酶切鉴定 ,将获得的 4个非重复克隆并进行序列分析 .结果 :筛选出 4个与MINT F2相互作用的蛋白 ,它们分别是 :6 7ku层黏连蛋白受体 ,,2个 1 6S核糖体RNA ,精氨酰 tRNA合成酶 .结论 :成功筛选出与MINT F2相互作用的蛋白 。

AIM: To identify the function and mechanism of nuclear matrix protein MINT (Msx2 interacting nuclear target protein). METHODS: Yeast two hybrid assay was used to screen proteins that interact with a fragment of MINT F2 (365-1084aa). From 1.0×10 8 yeast clones transformed with the bait plasmid and a cDNA library of 9 dpc mouse embryo,128 were positive for nutritional screening and β galactosidase assay. Restriction digestion showed 4 independent positive clones and they were analyzed by DNA sequencing, and revealed that they represent correctly fused cDNA fragments. RESULTS: Screened 4 proteins that interacting with MINT F2, which are 67 ku laminin receptor, two of 16 S ribosomal RNA, Arginyl tRNA synthetase. CONCLUSION: Succeed screening of interacting with MINT F2 proteins, and made the basement for further study of the function and mechanism of MINT.