كلونينگ cDNA پروتئين پراكسيزومي موش و بررسي جاي گيري درون سلولي آن

Mouse Peroxisomal Protein cDNA Cloning and Characterization of its Intraclleular Localization

Objective: The aim of this study was to clone peroxisomal protein (PEP) cDNA in a mammalian expression vector in a chimeric cDNA type, with enhanced green fluorescent protein (EGFP) cDNA. To invest igate the intracellular localization of PEP protein linked to EGFP marker. the constructed plasmid was used for transfection into the chinese hamster ovary (CHO) cells . Materials and Methods: Total RNA was extracted from the heart tissue of an adult mouse . PEP cDNA was constructed using reverse transcriptase and was amplified with specific primers covering the entire length of ORF. RT-PCR products containing PEP cDNA were treated by enzymatic digestion and inserted into the pEGFP-C1 downstream of EGFP cDNA and were used for transformation into bacterial competent cells. The positive colonies which showed inserted PEP cDNA were selected for plasmid preparations and additional analysis was performed to ensure that PEP eDNA was inserted properly. Finally, to confirm the intracellular localization of EGFP-PEP. CHO cells were transfected with the constructed plasmid. Results : Our results confirmed amplification and cloning of the expected product. PEP cDNA encompasses 630 bp which encodes 209 amino acid residues . Bioinformalics analyses have shown the presence of a fibronectin type III domain (31-114 a.a.) and two hydrophobic domains (12-32 a.a. and 152-169 a.a., respectively) . Because of the presence of serine, Lysine, leucine (SKI) in the C-terminal of the related protein , transfection data showed peroxisomal localization of PEP as was similar to the catalase . Conclusion: Taken together these data showed that PEP is a peroxisomal protein. However the importance of its fibronectin type 111 and two hydrophobic domains should be assessed by further experiments.