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[Construction, expression and identification of recombinant plasmid encoding bifunctional protein sflk1-IFN-gamma].

OBJECTIVE
To construct, express and identify the recombinant plasmid pcDNA3.1(+)/sflk1-IFN-gamma encoding bifunctional protein sflk1-IFN-gamma (soluble fetal liver kinase 1 and interferon-gamma).


METHODS
sflk1 and IFN-gamma gene fragments were cloned by RT-PCR, and then inserted into pcDNA3.1(+) plasmid between BamHI-EcoRI and XhoI-XbaI restriction sites to form the recombinant plasmid pcDNA3.1(+)/sflk1-IFN-gamma. The recombinant sflk1-IFN-gamma transiently expressed in COS-7 cells was detected by ELISA and Western blotting. Bioactivities of sflk1-IFN-gamma fusion protein were identified by proliferation inhibition assay with H5V cells and NK activity assay.


RESULTS
pcDNA3.1(+)/sflk1-IFN-gamma can be effectively expressed in COS-7 cells. Concentrations of sflk1 and IFN-gamma in culture supernatants of pcDNA3.1(+)/sflk1-IFN-gamma transfected COS-7 cells were (20.85+/-2.48) ng/ml and (1.08+/-0.09) ng/ml, respectively. Western blotting showed that the molecular weight of sflk1-IFN-gamma fusion protein was about 130 kDa, while that of sflk1 was 115 kDa. The supernatants of transfected cells significantly inhibited the proliferation of H5V cells stimulated by mouse VEGF 164 and enhanced the NK activity of splenocytes, demonstrating that sflk1-IFN-gamma fusion protein possessed the bioactivities of both sflk1 and IFN-gamma.


CONCLUSION
The constructed plasmid pcDNA3.1(+)/sflk1-IFN-gamma can be effectively expressed in eukaryotes. The expressed sflk1-IFN-gamma fusion protein has the biological activities of both sflk1 and IFN-gamma.