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OALib Journal期刊
ISSN: 2333-9721
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Construction of recombinant retroviral vector carrying porcine interferon-gamma and its expression in porcine kidney cells (PK-15)
携带猪γ干扰素基因逆转录病毒载体的构建及其在猪肾细胞(PK-15)中的表达

Keywords: Porcine interferon-gamma,Retroviral vector,PA317,PK-15,Expression,Antiviral activity,VSV,FMDV
猪γ干扰素
,逆转录病毒,PA317细胞,PK-15细胞,表达,抗病毒活性,水泡性口炎病毒,口蹄疫病毒

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Abstract:

Porcine interferon-gamma (PoIFN-gamma) of Chinese local brand, Meishan porcine, was cloned and inserted into retroviral vector pLXSN (neo r) . Using Lipofectamine, this recombinant plasmid was transfected into retroviral packing cell line, PA317 cells. These transfected cells were selected by DMEM containing 400microg/mL G418 for one week. RNA was extracted from the supernatant of these selected PA317 cells and the PoIFN-gamma gene could be amplified by RT-PCR. Pocine kidney cells and PK-15 cells were infected by the supernatant and were selected by 400 microg/mL, 600 microg/mL and 800 microg/mL G418, respectively. Those PK-15 cells were detected by indirect immunofluorescence assay and it was found that PoIFN-gamma 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/10(6) cells. In addition, the effect of rPoIFNgamma-anti-FMDV was determined using cytopathic effect inhibition. The results indicate that PoIFN-gamma 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-gamma with natural antiviral bioactivity and can inhibit VSV and FMDV.

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