Construction of recombinant retroviral vector carrying porcine interferon-gamma and its expression in porcine kidney cells (PK-15)

Abstract:

Porcine interferon-gamma (PoIFN-γ) of Chinese local brand, Meishan porcine, was cloned and inserted into retroviral vector pLXSN (neo^r). Using LipofectamineTM, this recombinant plasmid was transfected into retroviral packing cell line, PA317 cells. These transfected cells were selected by DMEM containing 400μg/mL G418 for one week. RNA was extracted from the supernatant of these selected PA317 cells and the PoIFN-γ gene could be amplified by RT-PCR. Pocine kidney cells and PK-15 cells were infected by the supernatant and were selected by 400μg/mL, 600μg/mL and 800μg/mL G418, respectively. Those PK-15 cells were detected by indirect immunofluorescence assay and it was found that PoIFN-γ mainly anchored in cellular membrane. The supernatant of the selected PK-15 was tested for the antiviral bioactivity after 48 hours of passage. The anti-VSV (vesicular stomatitis virus) activity in MDBK (bovine kidney cell) was 1200IU/106cells. In addition, the effect of rPoIFNγ-anti-FMDV was determined using cytopathic effect inhibition. The results indicate that PoIFN-γ has been inserted into retroviral vector and recombinant retrovirus has been successfully packaged in PA317 cells. Furthermore, this retrovirus can infect PK-15 cells and express PoIFN-γ with natural antiviral bioactivity and can inhibit VSV and FMDV.

Key words:Porcine interferon-gamma; Retroviral vector; PA317; PK-15; Expression; Antiviral activity; VSV; FMDV