Construction of Shuttle Plasmid for Co-expression of Ipr1 and GFP Genes in Human Lung Cancer Cells

Objective To construct a shuttle plasmid for co-expression of intracellular pathogen resistance gene 1(Ipr1)and green fluorescent protein(GFP)gene in human lung cancer A549 cells. Methods Ipr1 and GFP genes were amplified from plasmids pEGFP-C1-Ipr1 and pEGFP-C1 by PCR respectively. The amplified Ipr1 and GFP genes as well as mycobacterium replicon OriM were cloned into eukaryotic co-expression vector pBudCE4.1, and the constructed shuttle plasmid pBud-GFP-OriM-Ipr1 was transfected to A549 cells in mediation of liposome. The expression of GFP was observed by fluorescent microscopy, and that of Ipr1 protein was determined by immunohistochemical assay. Results Both restriction analysis and sequencing proved that shuttle plasmid pBudGFP-OriM-Ipr1 was constructed correctly. Expressed GFP was observed by fluorescent microscopy in transfected A549 cells. Immunohistochemical assay proved that Ipr1 protein was expressed in transfected A549 cells and located in cell nucleus. Conclusion A shuttle plasmid for eukaryotic co-expression of Ipr1 and GFP genes was successfully constructed, which laid a foundation of further study on function of Ipr1 against tuberculosis.