<p><b>OBJECTIVE</b>To construct a novel immunogene
therapeutic plasmid that expresses
human interleukin-12 (
IL-12),
granulocyte-
macrophage colony stimulating factor (
GM-CSF) and B7.1 and observe its expression in vivo and
in vitro.</p><p><b>
METHODS</b>
Human IL-12 gene fragment was cloned into the upper
stream of IRES
gene in the previously constructed
plasmid pVAX-IRES-
GM-CSF-B7.1, and the positive recombinant
plasmid pVAX-
IL-12-GB was transfected into
293T cells via Lipofectamine 2000. The expressions of
IL-12 and
GM-CSF-B7.1
mRNA and
proteins in the transfected
cells were assayed by RT-PCR and
ELISA, and B7.1 expression was tested by
fluorescence-activated cell sorting and
immunofluorescence assay. The
plasmid pVAX-
IL-12-GB was delivered into
mouse muscle by
electroporation, and the expression of
IL-12 in the
muscle tissue was identified by
immunohistochemistry.</p><p><b>RESULTS</b>
Enzyme digestion,
PCR and
sequence analysis all confirmed successful
construction of the recombinant
plasmid pVAX-
IL-12-GB.
IL-12,
GM-CSF and B7.1 expressions were all detected in transfected
293T cells, and the expression of
IL-12 was also detected in the transfected
mouse muscular
tissues.</p><p><b>CONCLUSION</b>A novel anti-
tumor immunogene
vaccine constructed can be expressed both in vivo and
in vitro, which facilitates further studies of
tumor immunogene
therapy.</p>