Transfection of PC12 cell line by pEGFPNI- NT4 and protein expression analysis

Cloning and expression in the cells is one of the most important basic techniques to study a specific protein in molecular biology. Among gene transfer methods, non-viral methods are less expensive, easier and safer to be implemented. NT4 is an important gene with miscellaneous clinical applications. This gene is a neurotrophic that induce survival, development and function of neurons as well as inducing differentiation of progenitor cells to form neurons. The aim of this study was to construct and transfect NT4 gene, in order to use it in research and gene therapy. NT4 gene was subcloned in plasmid pcmvsport6, using PCR; then it was transformed in Escherichia coli bacteria strain DH5-alpha. The recombinant gene was extracted and restriction enzymes by NOTI, SAII. The fragment gene was legated into the PEGFP-N1 plasmid. Then by restriction enzymes HINDIII, the recombinant was detected through gel electrophosis. In order to ensure that recombinant gene is correct, it was sended for sequencing. Cell culture was prepared and the recombinant plasmid was transfected. SDS page and western blotting were used for detection of protein expression. Enzyme analysis showed that pcDNA had correct structure and sequencing, confirmed by 100% homology of the gene with reported alpha gene in Gene Bank. In the analysis of proteins isolated from transfected cells with SDS-PAGE, an approximately 45 kDa band was observed with monoclonal antibody which was confirmed by Western blot analysis. This plasmid is able to transform to Eukaryotic system and is suitable for evaluation of translation due to its proper structure