分泌蛋白特异性基因陷阱的设计与验证

Design and Efficacy of a Gene Trap System for Secretory Proteins

蛋白质分子向细胞外分泌的过程有赖于信号肽的介导 .基因陷阱是功能性基因克隆的一种有效方法 ,经典的基因陷阱以geo作为报告基因 ,对所克隆的基因类型没有选择性 .将绿脓杆菌外毒素、 2A序列、IL 2受体穿膜区以及新霉素磷酸转移酶的基因依次融合构建新型报告基因——— peo ,该报告基因能够特异性地甄别具有信号肽编码序列的基因 .为验证 peo对信号肽编码序列的特异性甄别作用 ,以 peo为报告基因 ,构建了 3种质粒载体 ,分别模拟用基因陷阱载体进行筛选时可能出现的 3种情况 ,通过转染HeLa细胞、G4 18筛选以及碱性磷酸酶检测 ,证明 peo能够有效地区分信号肽和非信号肽编码序列 ,以 peo为报告基因改造的基因陷阱载体可以用于分泌蛋白基因的筛选 .

The secretion of proteins depends on signal peptide. Gene trap is a powerful tool for functional gene identification and characterization. Classical gene trap employs geo as the reporter, and gives no discrimination on the types of trapped genes. A new reporter gene named peo was obtained by in turn fusing Exotoxin A of Pseudomonas aeruginosa, 2A sequence, transmembrane region of IL-2 receptor and neomycin phosphotransferase genes, Peo is able to report genes encoding secretory proteins specifically. To prove peo's specificity for secretory proteins, three plasmid vectors, representing three types of sequences possibly trapped by gene trap vector during screening, were constructed based on peo reporter. After transfecting HeLa cells with these plasmids, followed by G418 selection and PLAP staining, it is shown that the new vector could distinguish secretory proteins from others, and the gene trap vector taking peo as the reporter can trap secretory protein genes efficiently.