Objective: To construct the eukaryotic expression vector of TIMP-2 gene and to explore its expression in human ameloblastoma cell in vitro.
Methods: The aimed gene fragment was obtained by RT-PCR. And then, molecmicrolar cloning technology and enzyme digestion were used to connect the gene with the plasmid PcDNA3.1(+), which can be expressed in eukaryotic cells and a report gene: green fluorescent protein gene (GFP) was already existed in the plasmid. We named the eukaryotic expression vector, which contended our aimed gene TIMP-2 as well as report gene GFP, PcDNA3.1(+)/GFP-TIMP-2. The vector was identified by PCR analysis, EcoR I and Xho I restriction analysis and Sequence analysis. After the PcDNA3.1(+)/GFP-TIMP-2 was transfected into cultured human ameloblastoma cell, RT-PCR and Flow Cytometry (FCM) and Microscope wre respectively performed to evaluate the effect of transfection and expression.
Results: The constructed vector PcDNA3.1(+)/GFP-TIMP-2 was proved correct by enzyme digestion and sequencing analysis. After PcDNA3.1(+)/GFP-TIMP-2 was trasnfected into cultured human ameloblastoma cell, the rate of transfection is 47.6% (Analysis report of FCM), the green fluorescence was found in plasm (observed with fluo-microwave), the expression of TIMP-2 mRNA was elevated 2.4 times compared with the control group.
Conclusions: PcDNA3.1(+)/GFP-TIMP-2 was successfully constructed and it could be transfected into cultured human ameloblastoma cell. It may be benefit to further study of the relationship between the TIMP-2 gene and the behaviour of ameloblastoma.