[Construction of recombinant retroviral vector carrying porcine interferon-gamma and its expression in porcine kidney cells (PK-15)].
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.
Yao
Qing-xia
QX
Laboratory of Animal Virology , College of Animal Science and Veterinary Medicine, Huazhong Agricultural University , Wuhan 430070, China. yaoqingxia@sohu.com
Liu
Xin-wen
XW
Qian
Ping
P
Guo
Dong-chun
DC
Chen
Huan-chun
HC
D000818:Animals; D019084:Fluorescent Antibody Technique, Indirect; D029544:Foot-and-Mouth Disease Virus / Q000187:drug effects; D005822:Genetic Vectors; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism*; D011994:Recombinant Proteins; D012190:Retroviridae / Q000235:genetics*; D013552:Swine; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects
D011994:Recombinant Proteins; D007371:Interferon-gamma