Objective   To construct an eukaryotic co-expression plasmid pBM9 carrying human granulysin active peptide and murine IL-12 and determine its expression.    Methods   The primer pairs including granulysin leader peptide DNA sequence were designed to amplify granulysin from the plasmid pZM03 carrying granulysin gene by polymerase chain reaction (PCR). PCR product was directly cloned into an eukaryotic co-expression plasmid pBudCE4.1 to construct plasmid pBudCE4.1-S9K. pBudCE4.1-S9K plasmid was identified by DNA sequencing. Murine IL-12 gene was subcloned into pBudCE4.1-S9K to construct eukaryotic co-expression plasmid pBudCE4.1-S9K/mIL-12. Mycobacteria replicon Orim from plasmid pZM03 was subcloned into NotⅠsite of pBudCE4.1-S9K/mIL-12 to construct eukaryotic co-expression shuttle plasmid pBM9. pBM9 was tansfected into RAW264.7 cells. RT-PCR was used to detect the mRNA expressions of granulysin and IL-12. The protein expression of them were observed by immunocytochemical method and ELISA respectively.    Results   RT-PCR identified the expressions of granulysin and mIL-12 in transfected cells and culture supernatant. Immunocytochemical method and ELISA verified the protein expressions.    Conclusion   The recombinant shuttle plasmid pBM9 is successfully constructed and expressed in vitro

which laid a foundation of gene therapy for tumors with granulysin and mIL-12.