Construction of eukaryotic expression vectors following connective tissue growth factor gene cloning in mice

BACKGROUND: There is strong evidence demonstrated that connective tissue growth factor (CTGF) showed higher expression in vascular smooth muscle cells (VSMC) with artherosclerosis, therefore, construction eukaryotic expression vector of connective is the basis for research the action mechanism of tissue growth factor expression. OBJECTIVE: To clone mice CTGF gene and construct its eukaryotic expression vector, in addition, to investigate its transient expression in VSMC. DESIGN, TIME AND SETTING: A single sample observation was performed at the High Educational Key Laboratory of Molecular Cell Biology and Drug Research, Liaoning Medical University from September 2007 to April 2008. MATERIALS: A total of 10 Kunming mice, with 1-month-old. The expression vector of pcDNA3.1(+)were preserved in Laboratory of Molecular Biology Department of Liaoning Medical University. METHODS: Using total RNA of mus kidney as template, amplified the fragment of mice CTGF gene by RT-PCR, and then cloned into pMD18-T vector. The eukaryotic expression vector pcDNA3.1 (+)-CTGF was constructed by ligating mi e CTGF gene with pcDNA3.1(+) vector. MAIN OUTCOME MEASURES: The recombinant pcDNA3.1 (+)-CTGF was identified by double enzymes digestion. Then tranfected pcDNA3.1 (+)-CTGF into VSMC. The expression of CTGF was measured by Western blot. RESULTS: The restriction endonuclease digestion and sequencing identified that the eukaryotic expression vector caning CTGF gene was constructed successfully, and CTGF protein was highly expressed following transfe ted into VSMC. CONCLUSION: The recombinant pcDNA3.1(+)-CTGF have been achieved successfully, which can express in VSMC.