Cloning of Human Survivin Gene Into Pet28a Bacterial Vector

Background and aim: apoptosis is a form of programmed cell death leading to the elimination of cell without releasing harmful substances into the surrounding area. inhibitor of apoptosis are a family of proteins that mainly block programmed cell death. the human iap family consist of 8 members. survivin is a smallest well-known inhibitor of apoptosis proteins family member. survivin overexpression occurs in many different cancer types but not in the normal tissue except embryonic tissue. survivin may be used as a new marker to stratify cancerous patients for more optimal treatment modalities, or can be a promising new target for therapy. in this research, we cloned human survivin gene into pet28a bacterial vector.methods: specific forward and reverse primers were designed. pcr was performed and survivin gene amplified by specific primers and tempelet. pcr product of survivin and pet28a vector were digested by hindiii/nhei restriction enzymes and survivin was ligated into the hindiii/nhei digested/dephosphorylated pet28a vector. then, the ligation product was transformed into the e.coli dh5a competent cells and screened by antibiotic selection marker (kanamycine).results: positive colonies were selected by colony pcr and screened by double digestion of isolated plasmid. one positive colony was sequenced and confirmed the inserted dna.conclusion: human survivin gene was cloned in pet28a vector. after validation by sequencing, pet28a/survivin product was transformed into the e.coli bl21(de3) competent cells to study recombinant protein expression.