Designing and Constructing Episomal Plasmid Based on Mutated Large T Antigen Simian Virus 40

The aim of this study was to design and construct a plasmid containing mutated large T antigen and replication origin of SV40 to develop a safe vector which can be recognized and replicated episomally by trans elements of eukaryotic cells without any interaction with P53 and retinoblastoma. Therefore، it can have a safe application in gene therapy and recombinant protein production is possible. Suitable mutants for creating safe large T antigen were analyzed by MODELLER software and finally three appropriate structures were selected. Desired mutations were created in the nucleotide sequence of large T antigen by PCR method. Mutated large T antigen gene was cloned in the IRES2-EGFP. All clones were analyzed by PCR، restriction analysis and sequencing. HEK293 and CHO cell lines were transfected by final construct and transfected cells were observed by fluorescent microscope for 40 days. Plasmid and genomic DNA were extracted from remained cells and overlap PCRs performed on them to confirm their circularity. This plasmid، contains mutated large T antigen and replication origin of SV40، can be replicated in eukaryotic cells and then can be used in gene therapy and recombinant protein production with high safety. The results of PCR، restriction analysis and sequencing confirm the authenticity of construct. The transfection of HEK293 and CHO cell lines showed replication of constructed plasmid in them.