Isolation and Cloning of MAGEA4 human gene into pRUF expression plasmid

Aim and Cancer/testis (CT) antigens are a category of tumor antigens that are typically expressed only in the human germ line, and in several types of tumor. MAGEA4 is one member cancer testis antigen family that has relation with other tumors. Since the biological function of MAGEA4 is unclear, the aim of the present study was amplification and cloning of the gene in the expression vector to produce recombinant protein that expresses MAGEA4. Using PCR specific primers including restriction site, MAGEA4 was amplified. The purified PCR products were ligated between the BamH1 and Xho1 sites of pTZ57/R cloning vector and transformed into Escherichia coli Top10 strain and screened by IPTG and X-Gal. The correct orientation of MAGEA4 fragment was identified by restriction enzyme analysis and sequencing of constructed plasmid. Then sub cloning was carried out within pRUF expression vector with the same restriction site. The final confirmation was performed using colony PCR, double digesting and sequence analysis. Therefore the MAGEA4 gene was cloned into the restriction site of pRUF. This study is an important step for producing recombinant proteins and is used to find the function of this gene for therapeutic targets.