脑胶质瘤相关新基因PKIβ的表达与蛋白质性质的研究

Expression and characterizations of a novel full-length gene PKIβ related to human glioma

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1条基因进行了克隆和表达。方法抽提正常成人脑组织与脑胶质瘤组织中的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436F11克隆子进行Northern印迹，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印迹证实436F11基因在人正常脑组织中高表达，而在人脑胶质瘤中低表达。BLASTn和BLASTx分析显示，436F11基因为全长新基因，共编码78个氨基酸，其理论相对分子质量为8468等电点为4．69，与鼠PKIβ69％同源，命名为人PKIβ。并在大肠杆菌中得到了PKIβ较高的表达蛋白，经纯化，在SDS—PAGE胶上获得了1条清晰的条带。氨基酸测序和分子量测定与生物信息学结果完全一致。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。人PKIβ可能是与人脑胶质瘤形成有关的1条全长新基因，这为脑胶质瘤的基因治疗提供了一条新思路。

Objective To obtain differentially expressed genes related to human glioma using cDNA mieroarray and to characterization of one novel full-length gene. Method Four samples of human glioma samples, 1 fetal brain tissue sample, and 2 normal brain tissue samples were used to extract the total RNA, and the mRNA was used to make probes. After hybridization and washing procedure, the products of hybridization were scanned using computer system. One gene, named 436F11 clone, was subsequently analyzed by Northern blotting, bioinformatics, and protein expression. Result Fifteen differentially expressed new genes related to human glioma were obtained through four times of hybridization and scanning. Northern blotting confirmed that over-expression of 436F11 gene in the normal human brain tissue and lowexpression in the human glioma tissues. The analysis of BLASTn and BLASTx showed that the clone of 436F11 was a novel full-length gene coding 78 amino acids of protein with a theoretical relative molecular weight of 8648 and an isoelectric point of 4.69 and that it was 60% identical to mouse PKIβ amino acid, so it was called human PKIβ gene. After it was transfected into Escherichia . coli, higher-expressed protein of PKIβ was obtained which yielded a major clear band on an SDS-PAGE gel after purification. The products obtained from amino acid sequencing and molecular weight detection were exactly the same as the products performed by bioinformatic analysis. Conclusion cDNA microarray technology can be successfully applied to identify differentially expressed genes. PKIβ may be a novel full-length gene related to human glioma and mav nrovide a new wav to gene theraov of glioma.