Yao Qing-xia, Liu Xin-wen, Qian Ping, Guo Dong-chun, Chen Huan-chun
Laboratory of Animal Virology , College of Animal Science and Veterinary Medicine, Huazhong Agricultural University , Wuhan 430070, China.
Wei Sheng Wu Xue Bao. 2007 Feb;47(1):141-4.
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.
对中国本土品牌梅山猪的猪干扰素-γ（PoIFN-γ）进行克隆，并将其插入逆转录病毒载体pLXSN（新霉素抗性）。使用脂质体转染试剂，将该重组质粒转染到逆转录病毒包装细胞系PA317细胞中。用含有400μg/mL G418的DMEM培养基对这些转染细胞进行筛选，持续一周。从这些筛选后的PA317细胞的上清液中提取RNA，通过RT-PCR扩增PoIFN-γ基因。用该上清液感染猪肾细胞和PK-15细胞，分别用400μg/mL、600μg/mL和800μg/mL G418进行筛选。通过间接免疫荧光法检测那些PK-15细胞，发现PoIFN-γ主要锚定在细胞膜上。对筛选后的PK-15细胞的上清液传代48小时后检测其抗病毒生物活性。在牛肾细胞（MDBK）中的抗水泡性口炎病毒（VSV）活性为1200IU/10⁶细胞。此外，使用细胞病变效应抑制法测定重组猪干扰素-γ抗口蹄疫病毒（FMDV）的效果。结果表明，PoIFN-γ已插入逆转录病毒载体，重组逆转录病毒已在PA317细胞中成功包装。此外，这种逆转录病毒可以感染PK-15细胞并表达具有天然抗病毒生物活性的PoIFN-γ，并且可以抑制VSV和FMDV。
