快速检测核糖体失活蛋白活性质粒的构建及应用  

Construction of a Plasmid to Detect the N-glycosidase Activity of Ribosome Inactivating Proteins

作　　者：吕彦[1] 崔晓东[1] 李玉英[1] 王转花[1] 

机构地区：[1]山西大学生物技术研究所,化学生物学与分子工程教育部重点实验室,太原030006

出　　处：《中国生物化学与分子生物学报》2016年第1期49-55,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.31171659/31300653)项目;山西省科技平台项目(No.2014091028)资助

摘　　要：核糖体失活蛋白专一地断裂28S rRNA第4 324位的腺嘌呤与核糖之间的N-糖苷键,具有特异破坏核糖体的结构,抑制蛋白质生物合成的功能。核糖体失活蛋白在医疗方面有极大的应用价值。为了能简单快速筛选出核糖体失活蛋白,本实验构建了一种包含核糖体失活蛋白识别位点的双荧光素酶质粒psiCHECK^(TM)-2-F28RNA。用具有N-糖苷酶活性的苦荞凝集素(tartary buckwheat lectin,TBL)作用于psiCHECK^(TM)-2-F28RNA质粒,电泳检测发现,TBL可以将质粒DNA由超螺旋型切割为缺刻型。将psiCHECK^(TM)-2-F28RNA转染HCT116细胞,发现海肾/萤火虫荧光比值也明显降低,表明构建的质粒可以用于检测核糖体失活蛋白对细胞的毒性作用。当将psiCHECK^(TM)-2-F28RNA中的GAGA序列中腺嘌呤分别突变后进行同样实验,确定该质粒中的GAGA为核糖体失活蛋白的识别位点。进一步构建包含GAGA特征序列的Wnt1-3'UTR区的质粒psiCHECK^(TM)-2-Wnt1-3'UTR,实验也发现,在胞外和胞内TBL与psiCHECK^(TM)-2-Wnt1-3'UTR都具有相互作用,表明细胞内具有GAGA序列的m RNA也可能成为核糖体失活蛋白的靶点。选用几种食源性作物中提取的蛋白质,分别与psiCHECK^(TM)-2-F28RNA作用,进行体外检测,结果显示,该质粒能快速地筛选来源于不同生物的核糖体失活蛋白。这些结果表明,本实验构建的psiCHECK^(TM)-2-F28RNA质粒,可用于核糖体失活蛋白的快速筛选和酶活性鉴定。Ribosome inactivating proteins（ RIPs） inhibit the protein translation and are known to hydrolyze the N-glycosidic bond between adenine 4 324 and the ribose of 28 S r RNAs. A recombinant dual luciferase plasmid containing the RIP recognition site named psiCHECKTM-2-F28 RNA was constructed.Tartary buckwheat lectin（ TBL）,a N-glycosidase inhibitor was used to incubated with psiCHECKTM-2-F28 RNA before agarose gel electrophoresis. It showed that the plasmid was changed into nicked type from supercoiled type. When transfected into HCT116 cells,decreased fluorescence ratio（ renilla / firefly） was observed. The results indicated that our plasmid could be utilized to detect cellular toxicity of RIPs. We verified that the GAGA sequences was the site of a potential RIP target by site-directed,mutagenesis ApsiCHECKTM-2-Wnt1 3＇ UTR plasmid was constructed and was tested in electrophoresis and dual luciferase assay. These findings suggested that psiCHECKTM-2-F28 RNA can be used for screening and assaying enzymatic activity of RIPs.

关 键 词：核糖体失活蛋白 苦荞凝集素 28SrRNA GAGA特征序列 MRNA 

分 类 号：Q51[生物学—生物化学] Q71