Molecular cloning, expression and characterization of rat tumor necrosis factor-α as potent anti-tumor candidate

Tumor Necrosis factor (TNF), a pleiotropic cytokine, has been shown to have direct anti-tumor activity killing tumor cells while sparing normal cells. In the present study, we amplified and cloned the rat TNF-α into pcDNA3.1 (+) eukaryotic expression vector for its future use as genetic adjuvant in the treatment of chemically induced skin tumor model (Wistar rats). The recombinant pcDNA.rat.tnf was confirmed by restriction enzyme digestion and nucleotide sequencing. The in-vitro expression of recombinant pcDNA.rat.tnf was checked both at transcriptional and translational levels in transfected HeLa cells by RT-PCR, flow cytometry and immunofluorescence. Further, the characterization of in-vivo expression of the protein was carried out by sandwich ELISA using serum from pcDNA.rat.tnf treated Wistar rats. We report successful cloning and expression of rat TNF-α in a eukaryotic expression vector pcDNA3.1 (+). The construct pcDNA.rat.tnf can now be used as an anti-tumor agent alone or along with other anti-tumor therapies as an adjuvant.