Construction and expression of human akt1 gene

    SHEN Lan, LI Xia, ZHANG Ziª²Feng, SU Jin, LIU Xinª²Ping, YAO Liª²Bo

    Department of Biochemstry and Molecular Biology, School of Basic Medicine, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To construct the eukaryotic expressing vectors of human akt1 and Nª²terminally myristoylation signalª²attached akt1 (myrª²akt1) and to test their expression in African green monkey kidney cell line COSª²7. METHODS: Human akt1 and myrª²akt1 genes tagged with flag were amplified from human dental pulp mRNA by RTª²PCR. The products were cloned into pMDª²18T vector, sequenced and then subcloned into eukaryotic expressing vector pcDNA3.1(+). The recombined vectors were then transfected into COSª²7 cells with lipofectin to detect the expression of akt1 and myrª²akt1 by Western blot. The cell cycle profiles of COSª²7 cells were analyzed by flow cytometry (FCM). RESULTS: The eukaryotic expressing vectors containing human akt1 and myrª²akt1 were successfully constructed and their expression in COSª²7 cells could be detected. AKT1 overexpression and activation showed little effect on the cell cycle of COSª²7 by cell cycle profiles. CONCLUSION: The eukaryotic expressing vectors containing human akt1 and myrª²akt1 are constructed and they can express flagª²akt1 and flagª²myrª²akt1 fused proteins correctly in COSª²7 cells. But the regulation of cell cycle by AKT1 has to be further investigated. ......