【作者】 郭晓旭；

【导师】 王兴智； 朱筱娟；

【作者基本信息】 东北师范大学 ， 遗传学， 2010， 硕士

【摘要】 酵母双杂交系统是目前研究蛋白质相互作用的重要手段之一,此方法具有高灵敏度、相对简单、快捷及不经蛋白纯化即可获得蛋白编码区的优点。近年来,酵母双杂交技术在研究蛋白质相互作用、筛选新蛋白、研究蛋白功能等方面发挥了重要作用。肌球蛋白X(Myosin X, Myo X)是仅表达于脊椎动物中的非传统肌球蛋白,其尾部的MyTH4和FERM结构域可与多种功能蛋白相结合。这些蛋白与丝足的形成、神经元的突起生长与投射、内皮细胞的迁移、细胞的粘附及有丝分裂中纺锤体的形成等过程密切相关。因此Myo X在脊椎动物细胞中发挥了重要作用。为了进一步深入的研究Myo X在脊椎动物体内的作用,本研究利用酵母双杂交技术,以Myo X的尾部结构域中的MyTH4和FERM结构为诱饵,对人胎脑cDNA文库进行了筛选。实验经Leu、Trp、His、Ade四种氨基酸营养缺陷型培养基筛选以及β-半乳糖苷酶活性分析,共获得112个阳性克隆,经过反复传代四种氨基酸营养缺陷型培养基筛选和β-半乳糖苷酶活性分析,去除一个酵母菌株中含有两个cDNA文库质粒的可能,再经过回交实验验证,最大程度的保证了酵母双杂交结果的准确性。从酵母菌株中提取质粒,转入大肠杆菌中,以氨苄青霉素(Amp)抗性筛选靶质粒,对部分阳性克隆的插入片段进行测序,在GENE BANK进行比对后分类,从分类结果可知与Myo X相互作用的蛋白涉及参与细胞迁移蛋白、细胞骨架蛋白、参与物质运输蛋白、参与基因表达调节的蛋白、参与信号转导蛋白等,为深入了解Myo X在生理上的新功能奠定基础。更多还原

【Abstract】 Yeast two-hybrid system is one of the most important methods to study protein-protein interaction. The system is sensitive, simple and rapid, which the sequence of target proteins can be obtained directly. For the last few years, yeast two-hybrid system is used to study protein-protein interaction, screen the novel proteins and study the function of protein.Myosin X is one of the members in unconventional myosin super-family and expresses at low concentrations in most vertebrate, which is relative to filopodial formation, neuronal growth, cell migration, cell adhesion and the spindle formation of mitosis. In this study, yeast two-hybrid technique was used to identify proteins interacting with Myo X. Here, the MyTH4 domain and FERM domain of Myo X tail are a bait to screen the human fetal brain cDNA library. Plating the cells on SD without histidine, tryptophan, leucine and adenine to assay the co-transformants to express the report genes and detect the expression of lac Z gene, a total of 112 positive clones were obtained, then, the positive clones were cultured in SD medium without histidine, tryptophan,leucine and adenine five times to remove the false clones. The backcross experiment was employed to ensure the results. The plasmid was extracted from yeast, and then it was transformed into E. coli and the target plasmid was obtained by selective culture. The cDNA segment of the positive clones were sequenced and compared with GENE BANK. The proteins screened in this study were probably involved in cell migration, cell adhesion, cystoskeleton rearrangement, protein transportation, regulation of gene expression and signal transduction.更多还原