《INMAP互作蛋白的筛选及验证》-毕业设计（论文）.docVIP

PAGE 26 INMAP互作蛋白的筛选及验证 摘　要 INMAP是本课题组发现的一种新的有丝分裂器蛋白，与纺锤体的形成和有丝分裂进程有关。为进一歩研究其生物学功能，我们运用酵母双杂交技术从HeLa细胞cDNA文库中筛选与其直接作用的蛋白质。首先利用PCR技术扩增INMAP的编码序列，并将其克隆至诱饵载体PGBKT7中，经测序鉴定后将其转化到酵母菌株AH109中。将含有诱饵载体的AH109与含有HeLa细胞cDNA文库的Y187菌株杂交，在含有X-a-gal的营养缺陷型培养基上进行选择性培养，筛选二倍体阳性酵母克隆，经回转实验和测序分析，验证得到4个与INMAP相互作用的蛋白质，它们分别是具有水解酶活性的FAM108B1、介导特定蛋白质自内质网向高尔基复合体（ER-to-Golgi）运输的MCFD2、核酸酶TATDN1和抑制端粒酶活性的YLPM1。这些互作蛋白的研究，对揭示INMAP的活性分子形成、跨细胞器运输或特定靶向作用和与有丝分裂器相关的功能机制必将具有重要的意义。此外，为进一歩验证此种相互作用，我们又进行了免疫共沉淀实验，结果发现……（实验正在进行中）。 关键词：INMAP，蛋白互作，酵母双杂交，免疫共沉淀 Screening and Identification of Proteins Interacting with INMAP Abstract INMAP, which is relevant to the formation of spindle and mitosis process, is a novel mitotic apparatus protein identified by our group. For the further studies on its biological function, a yeast two-bybrid assay was performed to screen proteins that interacts directly with INMAP from Pretransformed Normalized Human HeLa S3 Matchmaker? cDNA Library. INMAP code sequence obtained by PCR was cloned into bait vector pGBKT7. Being sequenced to confirm that the fusion construct is in-frame, pGBKT7-INMAP was transformed into yeast strain AH109 to generate a bait culture AH109[pGBKT7-INMAP]. The library was screened through a simple yeast mating procedure by mixing a concentrated bait culture with 1 ml of pretransformed library and incubating overnight, then plating the mated mixture on selective media and incubating at 30 °C till colonies appeared. Four positive colonies were confirmed by restreaking and retransformation in yeast. They are abhydrolase domain-containing protein FAM108B1, putative deoxyribonuclease TATDN1, nucleoside kinase YLPM1 with polynucleotide kinase/phosphohydrolase-like modular domains and MCFD2, which forms a specific cargo receptor with LMAN1 for the ER-to-Golgi transport of selected proteins. It must be of great significance to study on these interactions in revealing the formation of bioactive molecule, transport across organelles, specific targeting and the function