Construction and identification of eukaryotic expression vector of MAGE-3-HSP70 fusion gene

BACKGROUND: Vaccine-induced antitumor CD8+ T-cell responses are believed to play an important role in increasing resistance to carcinoma. Research shows that after uptake of fusion proteins of heat-shock protein 70 (HSP70) with tumor associated antigen into antigen presenting cells, processing and representation of heat-shock protein-chaperoned peptides on MHC class I molecules, a CD8+ cytotoxic T lymphocyte response is initiated. OBJECTIVE: To construct and identify eukaryotic expression vector of melanoma antigen gene 3 MAGE-3-HSP70. DESIGN, TIME AND SETTING: A single sample observation was completed at Key Laboratory of Nano-biotechnology, National Ministry of Health from June to December 2008. MATERIALS: The eukaryotic vector pcDNA3.1(-) was purchased from BD Company. The vector pINCY/MAGE-3 and pOTB7/HSP70 were purchased from Open Biosystems Company. DH5α were preserved at Key Laboratory of Nano-biotechnology, National Ministry of Public Health, China. METHODS: A pair of MAGE-3 primers containing Xho Ⅰ restriction site and HSP70 primers containing Kpn Ⅰ restriction site were designed. pINCY/MAGE-3 and pOTB7/HSP70 were used as template to amplify MAGE-3 and HSP70 gene by polymerase chain reaction (PCR). MAGE-3 upstream primer (containing Xho Ⅰ restriction site) and HSP70 downstream primer (containing Kpn Ⅰ restriction site) were used as template to amplify MAGE-3-HSP70 fusion gene fragments. The PCR products and empty plasmid pcDNA 3.1(-) were digested by XhoⅠ and Kpn Ⅰ, ligated, then transformed competence E.coli DH5α. Positive clones were identified by restriction endonuclease digestion and sequencing. MAIN OUTCOME MEASURES: The identification of pcDNA3.1(-)/MAGE-3-HSP70 fusion gene eukaryotic expression vector. RESULTS: Sequencing results showed that the MAGE-3 gene in the vector was in coincidence with the sequence registered in Gene Bank and the HSP70 had three synonymous mutations. CONCLUSION: MAGE-3-HSP70 fusion gene eukaryotic expression vector has been constructed successfully.