快速检测核糖体失活蛋白活性质粒的构建及应用

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psiCHECKTM-2-F28RNA。用具有N 糖苷酶活性的苦荞凝集素(tartary buckwheat lectin,TBL)作用于psiCHECKTM-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psiCHECKTM-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psiCHECKTM-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psiCHECKTM-2-Wnt1-3′UTR，实验也发现，在胞外和胞内TBL与psiCHECKTM-2-Wnt1-3′UTR都具有相互作用，表明细胞内具有GAGA序列的mRNA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psiCHECKTM-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psiCHECKTM-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。

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

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 28S rRNAs.A recombinant dual luciferase plasmid containing the RIP recognition site named psiCHECKTM-2-F28RNA was constructed. Tartary buckwheat lectin (TBL),a N-glycosidase inhibitor was used to incubated with psiCHECKTM-2-F28RNA 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 A psiCHECKTM-2-Wnt1 3′ UTR plasmid was constructed and was tested in electrophoresis and dual luciferase assay. These findings suggested that psiCHECKTM-2-F28RNA can be used for screening and assaying enzymatic activity of RIPs.