脑胶质瘤相关的全长新基因PPIL-3的克隆和蛋白质特性的研究

目的 运用基因芯片技术获取正常成人脑组织与人脑胶质瘤中差异表达的基因,并对其中一条与脑胶质瘤相关的新基因进行了克隆和表达的研究.方法 抽提正常成人脑组织与人脑胶质瘤组织中的mRNA来制备探针,经杂交、洗涤后,通过计算机观察二者表达谱的差异情况,对681F05克隆子进行了Northern blot,生物信息学分析和蛋白质的表达.结果 通过4次基因芯片筛选,获得15条与胶质瘤相关的新基因,经Northern blot证实681F05基因在人正常脑组织中低表达,而在人脑胶质瘤中高表达.BLASTn和BLASTx分析显示,它们编码蛋白与线虫Cyp-10蛋白同源性分别为52%和72%.cDNA序列分析发现这两个克隆是同一个基因(cyclophilin-like gene,PPIL3)的两个不同的剪切体(PPIL3a和PPIL3b).并在大肠杆菌中得到了PPIL3a和PPIL3b与GST较好表达的融合蛋白.PPIL3b蛋白具有依赖于Ca2+/Mg2+核酸酶活性,其核酸酶活性可被一定浓度的K+/Na+抑制.结论 基因芯片筛选正常脑组织与人脑胶质瘤差异表达的基因具有样品用量少、高质量、高速度、高敏感等特性.681F05基因可能是与人脑胶质瘤形成有关的一条全长新基因.

Isolation of one novel full-length gene PPIL-3 related to human glioma and characterization of this protein

Objective To obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. Method Total RNA was extracted from human glioma tissues and normal brain tissues,and mRNA was used to make probes. After hybridization and washing procedure, the results of hybridization were scanned using computer system. One gene named 681F05 clone was subsequently analyzed by northern blot, four times hybridizations and scanning. Northern blot analysis confirmed 681F05 clone was low-exprbioinformatics and protein expression. Results We obtain 15 differentially expressed genes to human glioma througession in human brain tissue and over-expression inhuman glioma tissues. The analysis of BLASTn and BLASTx showed that clone 681F05 we isolated was two cDNA clones encoding two novel proteins which show 52% and 72% identity to the eyelophilin isoform 10 of C. Elgans,respectively. Sequence analysis revealed these two cDNA clones are two different splicing variants of a novel cycophilin-like gene (PPIL3a and PPIL3b). In E. Coli. ,we got the more high-expressed fusion protein of PPIL3a combined with GST and PPIL3b combined with GST. Activity analysis of the recombinant PPIL3b protein shows that PPIL3b protein has nuclease activity. The nuclease activity of PPIL3b protein is stimulated by Ca2+ and/or Mg2+ and inhibited by K+/Na+. Conclusions We show that cDNA microarray technology can be successfully applied to identify differentially expressed genes. The novel full-length gene of human PPIL3 maybe correlate with forming human glioma.