Construction of Prokaryotic Expression Vector for Human HSP70 and MAGE-4 Epitope Genes

Objective To construct a prokaryotic expression vector for human heat shock protein 70(HSP70) and melanoma antigen-4(MAGE-4) epitope genes and identify the expressed product.Methods Under a condition of heat stress,amplify HSP70 gene from colon cancer cells by PCR.Analyze MAGE-4 antigen epitope by online predict software of protein secondary structure and amplify the epitope gene by PCR.Clone the amplified HSP70 and MAGE-4 epitope genes into pGEM-T easy vector and,after identification of the constructed recombinant plasmid by restriction analysis,subclone HSP70 gene into prokaryotic expression vector pET28a,and MAGE-4 gene downstream to HSP70.Transformed the constructed recombinant plasmid pET28a-HSP70-MAGE-4 to E.coli for expression under induction of IPTG.Identify the expressed product by SDS-PAGE and Western blot.Results Both PCR and DNA sequencing proved that recombinant plasmid pET28a-HSP70-MAGE-4 was correctly constructed.Both SDS-PAGE and Western blot showed positive bands consistent with those expected.Conclusion The prokaryotic expression vector for HSP70 and MAGE-4 epitope genes was successfully constructed,which provided a basis for the development of vaccine.