Sedlin突变体S124A对其与PAM14相互作用的影响  

Effect of Sedlin mutation S124A on interaction with PAM14

作　　者：汪云飞[1] 朱恒锐[1] 张庆慧[2] 邢昕[1] 武标[1] 刘晓颖[1] 范礼斌[1] 

机构地区：[1]安徽医科大学生物教研室,合肥230032 [2]昆山市第一人民医院检验科,昆山215300

出　　处：《安徽医科大学学报》2009年第1期10-13,共4页Acta Universitatis Medicinalis Anhui

基　　金：安徽医科大学博士科研基金(编号:XJ2005004)

摘　　要：目的构建Sedlin的点突变体,研究其与PAM14在酵母中的相互作用,寻找其相互作用的位点。方法用蛋白质在线序列分析软件CPHmodels预测Sedlin蛋白的磷酸化位点,可能的磷酸化位点主要有S2、S30、S119、S124、Y115,针对其中一个磷酸化位点(S124)设计定点突变引物;以pAS-Sedlin为模板,以两条含有突变位点的互补序列为引物进行PCR扩增,将得到的PCR产物用DpnⅠ酶切除去模板,消化产物转化大肠杆菌感受态DH5α,对得到的阳性克隆进行测序;将构建的定点突变质粒pAS-SedlinS与pACT2-PAM14共转化至酵母Y190中,并对生长至合适大小的克隆进行β-半乳糖苷酶活性分析。结果构建了pAS-Sedlin的定点突变质粒pAS-SedlinS,其与pACT2-PAM14共转克隆的β-半乳糖苷酶活性反应呈阳性,并且Sedlin S124A蛋白与PAM14蛋白的相互作用比野生型Sedlin蛋白与PAM14蛋白相互作用强。结论成功构建了Sedlin S124A,Sedlin蛋白的S124并不是其与PAM14在酵母中相互作用的特异性结合位点。Objective To construct the Sedlin S124A and define the binding site of this mutant for PAM14 in the yeast. Methods According to the program CPHm-odels, we predicted phosphorylation sites including S2, S30, S119, S124, Y115. Mutating S124 into A was our interest. So coding sequences of the Sedlin mutant were con- struced by modified method of Stratagene Site-Directed Mutagenesis Kit with pAS-Sedlin as a template. Positive clones had been selected with restriction enzyme fragment analysis and confirmed by nucleotide sequencing. Both of Sedlin（ or its mutant）and PAM14 were transformed into Y190 cells. The clones was then detected with β-galactosidase activity. Results The Sedlin S124A had been confirmed by sequence analysis. This mutant also interacted with PAM14 in yeast cells, and intensity of PAM14 interacting with the mutant was stronger than that of PAM14 interracting with wild type by β-galactosidase activity. This suggested the pohosphorylation of 5124 might affect cell growth. Conclusion The site-directed mutant Sedlin S124A has been successfully constructed. Sedlin S124A is not the specific site which interact with PAM14 in yeast cells.

关 键 词：蛋白质类/遗传学 酵母菌/遗传学 双杂交系统技术 骨骺 脊柱/畸形 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R341[医药卫生—基础医学]