生物信息学预测与实验验证相结合策略筛选鉴定新的人分泌蛋白基因

使用生物信息学预测结合实验验证的策略筛选鉴定人新的分泌蛋白基因。用SignalP、SOSUI、PSORT和BLAST等程序对UniProt蛋白数据库进行生物信息学分析 ,筛选出用于实验验证的 1 4个功能未知基因。采用RT PCR方法 ,克隆得到 1 4个基因的全长编码序列 ,并构建到真核表达载体pcDNA3.1 ( - ) Myc His质粒。采用蛋白质印迹与免疫荧光分析 ,检测到其中 7个基因的表达。除其中一个在细胞核表达外 ,其余 6个只在细胞质中表达 ;其中的 4个基因的表达产物在细胞培养液中可被检测到 ,鉴定为 4个新的分泌蛋白基因。

Isolate and Identify Human Novel Secreted Protein Genes with Combined Strategy of Bioinformatics Prediction and Experimental Confirmation

The strategy of bioinformatics prediction combined with experimental confirmation was used to isolate and identify novel potential human secreted protein genes.After bioinformatics prediction with SignalP,SOSUI,PSORT and BLAST programs on the UniProt Knowledgebase,14 genes were isolated for experimental identification.With RT-PCR amplification,all full coding sequences of these genes were cloned into mammalian cell expression plasmid,pcDNA3.1 (-)/Myc-His vector.7 genes were successfully expressed,judged by western blot and immunofluorescence analysis,in which one gene was expressed in nucleolus,the other six genes were expressed only in cytoplasm,and 4 expressed proteins of which were secreted into the culture medium and identified as novel secreted proteins.