Construction, expression and purification of recombinant HBcAg-MAGE-A3 therapeutic cancer vaccine

Objective: To construct and identify recombinant expression of a therapeutic tumor vaccine with HBcAg as a vector. Methods: PCR primers were designed according to the gene sequences of restriction enzyme sites of recombinant pKK233.2-hepatitis B core antigen (HBcAg) and melanoma-associated antigen 3 (MAGE-A3: 112120aa). The target fragment of MAGE-A3 was synthesized and cloned to pKK233.2-HBcAg expression vector. The recombinant pKK233.2-HBcAg-MAGE-A3 expression vector was identified by PCR detection followed by enzyme restriction and sequencing. The expression and purification of HBcAg-MAGE-A3 fusion protein at large scale were accomplished by crack bacterial precipitation through repeated freeze-thaw and ultracentrifugation. The recombinant protein vaccine was identified by western-blotting and dot blot. Results: The pKK233.2-HBcAg-MAGE-A3 expression vector was established. PCR and restriction enzyme digestion assays verified the size of the target fragment as predicted. The recombinant expression vector contained the full sequences of HBcAg and MAGE-A3 genes. Westernblotting and dot blotting analyses confirmed purity of recombinant HBcAg-MAGE-A3. Conclusion: Recombinant HBcAg-MAGE-A3 vectors and the methods for purifying therapeutic cancer vaccines were successfully established.