One novel full-length gene PKI related to human glioma

Abstract

Objective To obtain differentially expressed genes related to human glioma using cDNA microarray,and study the characterization of one novel full-length gene.Methods Total RNA was extracted from human g]ioma 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 436F11 clone,was subsequently analyzed by Northern blot,bioinformatics and protein expression.Results We obtain 15 differentially expressed genes related to human glioma through four times hybridizations and scanning.Northern blot analysis confirmed 436F11 clone was over-expressed in human brain tissue,and low-expressed in human glioma tissues,which was just the same as the result of in situ hybridization.The analysis of BLASTn and BLASTx showed that clone 436F11 was novel full-length gene.This gene coded 78 amino acids of protein,whose theoretical molecular weight was 8648 Da and PI was 4.69,while it was 69% identity to mouse PKI amino acid,so it was called human PK1 gene.After expression in E.coil,we got the more high-expressed protein of PKI which yielded a major clear band on the SDS-PAGE gel after purification.Human PKIβ protein(PKIβ-78 and PKIβ-71)with high purity inhibited phosphotransferase activity of C subunit in vitro.Conclusion cDNA microarray technology can be successfully applied to identify differentially expressed genes.The novel full-length gene of human PKI maybe correlate with formation of human glioma,and it could provide a new way to gene therapy of glioma. Key words: Glioma; cDNA microarray; Gene expression