Construction of expressing vectors including Mda-7 gene beside the RGD sequences for better tumor targeting

Background and objective: Up to now, many researches has performed to improve the anti-tumoral effect of mda-7 protein. The purpose of our research is to adjoin anti-tumoral effect of mda-7 and RGD peptide targeting to improve apoptosis induction in tumor site especially liver related onesMaterials and methods: Mda-7 gene with two different RGD sequencees amplified by PCR and then cloned into TA – cloning system. Colonies including these genes selected with Blue – White Screening, colony PCR and sequencing respectively. After that the genes digested and sub - cloned into expression vector following confirmation by colony PCR and sequencing. In addition, these construct transfected into 293 and Huh-7 cells for further expression assay. Protein expression then evaluated by RT-PCR and IF ( Immunofluorescence Assay ).Results and conclusion: Three different Mda-7 with RGD terminal peptide, were cloned correctly into expression vector and its expression evaluated with IF, RT-PCR, which was suitable. It was shown that these proteins were expressed and secreted into culture media and the rate of expression is considerable in 293 and Huh-7 cells. Theoretically RGD tagged Mda-7 would be able to induce apoptosis more strongly than the standard one therefore these new constructs has the potential forfuture researches.

Keywords:

RGD sequence , Mda-7 , apoptosis , cloning