全长新基因人核糖体蛋白L14．22在人脑胶质瘤中的研究与体外表达  

Isolation of one novel full-length gene related to human giioma and its expression in vitro

作　　者：祁震宇[1] 惠国桢[1] 李瑶[2] 周宗祥[2] 顾少华[2] 应康[2] 谢毅[2] 

机构地区：[1]苏州大学附属第一医院神经外科,215006 [2]复旦大学遗传工程国家重点开放实验室

出　　处：《中华实验外科杂志》2006年第5期554-556,共3页Chinese Journal of Experimental Surgery

摘　　要：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。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,通过计算机观察两者表达谱的差异情况,对507E08克隆子进行了Northern blot,生物信息学分析,蛋白表达和氨基酸测序。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507E08基因在人正常脑组织中低表达,而在人脑胶质瘤中高表达。原位杂交得到相同的结果。BLASTn和BLAST分析显示,507E08基因为全长新基因,长度为2 002 bp,共编码203个氨基酸。本基因命名为人核糖体蛋白L14．22。经原核表达,在SDS-PAGE胶上获得一条22×103的条带,氨基酸序列分析显示,结果N-端15个氨基酸与序列所示的N-端15个氨基酸残基完全相同。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。507E08基因可能是与人脑胶质瘤形成有关的一条全长新基因。Objective To obtain differentially expressed genes related to human glioma using cDNA mieroarray and the characterization of one novel full-length gene. Methods 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 507E08 clone was subsequently analyzed by Northern blot, bioinformatics, protein expression and amino acid sequencing. Results Fifteen differentially expressed genes to human glioma were obtained through 4 times hybridizations and scanning. Northern blot analysis confirmed 507E08 clone was low-expression in human brain tissue and over-expression in human glioma tissues. This result was just the same as that of in situ hybridization. The analysis of BLASTn and BLASTx showed that clone 507E08 was novel full-length gene coding 203 amino acid of protein, and itwas called human ribosomal protein 14.22 gene. After expression in E. coli, protein yielded a major band of apparent molecular mass 22 × 10^3 on an SDS-PAGE gel. Amino acid sequencing analysis of the recombinant L14.22 protein indicated that 15 amino acid sequences of N-termination was exactly same as the 15 amino acid residues of N-termination of L14.22 protein sequence. Conclusion cDNA mieroarray technology can be successfully applied to identify differentially expressed genes. The novel full-length gene of human ribosomal protein 14.22 may be correlate with pathogenesis of human glioma.

关 键 词：基因芯片 胶质瘤 基因表达 辐射杂交 

分 类 号：R739.4[医药卫生—肿瘤]