Cloning of human survivin gene into pET۲۸a 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 ۸ 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 pET۲۸a 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 pET۲۸a vector were digested by HindIII/NheI restriction enzymes and survivin was ligated into the HindIII/NheI digested/dephosphorylated pET۲۸a vector. Then, the ligation product was transformed into the E.coli DH۵a 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 pET۲۸a vector. After validation by sequencing, pET۲۸a/survivin product was transformed into the E.coli BL۲۱(DE۳) competent cells to study recombinant protein expression.

کلیدواژه ها:

Survivin ، Cloning ، pET۲۸a.