Cloning of full-length cDNA of mouse FABP3 gene,construction of eukaryotic expression vector and expression of the vector in COS-7 cells

Objective It had been found in previous gene expression microarray study that the gene of fatty acid binding protein 3 (FABP3) might contribute to the renal damage of patients with obesity related glomerulopathy. For further exploring the effect of FABP3 gene on podocyte lesion related obesity, the objective of present study was to construct the eukaryotic expression vector containing FABP3 gene, and to detect the expression of the vector in COS-7 cells. Methods The specific primers were designed according to the gene sequence published in GenBank, and the mouse FABP3 full-length cDNA was then cloned by RT-PCR. A 24bp FLAG coding sequence was also added to the 3' terminal of FABP3 cDNA using PCR. The open reading fragments of FABP3 and FABP3-flag were successfully inserted into pcDNA3.1 vector to construct eukaryotic expression vector pcDNA3.1-FABP3 and pcDNA3.1-FABP3-flag. The recombinant plasmids were transformed into the COS-7 cells for expression after the right sequence was confirmed by restriction enzymes analysis and DNA sequencing. The positive recombinants were transfected into COS-7 cells and the expression of FABP3 mRNA was detected by RT-PCR and the expression of FABP3 protein was assayed by immunofluorescence assay. Results The pcDNA3.1-FABP3 and pcDNA3.1-FABP3-flag treated with restriction enzymes EcoR Ⅰ and BamH Ⅰ produced 418bp and 442bp fragments, respectively, which were identified by sequencing as FABP3 and FABP3-flag. The object fragment of 278bp was amplified in COS-7/pcDNA3.1-FABP3 and COS-7/pcDNA3.1-FABP3-flag cells by RT-PCR. The special immunofluorescence staining was detected in the transfected cells. However, the same detections of COS-7/pcDNA3.1 cells gave negative results. Conclusion The eukaryotic expression vectors of pcDNA3.1-FABP3 and pcDNA3.1-FABP3-flag have been successfully constructed and expressed in COS-7 cells after the transfection.