Construction of Psicheck-2-Bcl2 in order to Perform luciferase Assay

Background and objective: After skin cancers, post-transplant Lymphoproliferativedisorders are the most common cancers among transplant patients. Most of the time, Epstein bar virus active genome has been found in tumor cells of these patients. Low expression of miR-15a-5p in any type of cancers has been demonstrated. This research aimed to study the effect of miR-15a-5p expression on one of the key factors, Bcl-2 gene, in this kind of malignancy. Material and methods: DNA was extracted from the whole blood of a healthy individual, amplified by PCR using specific 3′UTR region of Bcl-2 gene. PCR product was cloned in a cloning vector called PTZ57R/T. In a PTZ57R/T vector, Bcl-2 gene was prepared using xhoI and NotI restriction enzymes, and their restriction sites were placed at each end of the gene, at the same time. The gene was then subcloned in Psicheck-2 expression vector of named Psicheck-2-Bcl2 and conformed whit PCR and sequencing. Results: PCR and sequencing of recombinant Psicheck-2-Bcl-2 construct showed a 754bp fragment has been cloned. Conclusion: Since Bcl-2 gene has been overexpressed in PTLD cases, we hope that following miR-15-5p/Bcl-2 binding the Bcl-2 expression is down-regulated. In this study, luciferase assay was used to confirm the specific binding of these two.