[Expression of porcine gamma-interferon in recombinant baculovirus and determination of its antiviral activity].
The full-length porcine interferon gamma(PoIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into honor plasmid pFastBac 1 of Bac-To-Bac Baculovirus Expression System. These recombinant plasmids, pFastBac -PoIFN-gamma, were transformed into DH(10Bac) host bacteria to get recombinant shuttle plasmids, rBacmid-PoIFN-gamma. Recombinant baculovirus, rBac-PoIFN-gamma, was generated for expressing PoIFN-gamma, by transfecting rBacmid-PoIFN-gamma with Cellfectin Reagent into sf9 insect cells. The expression of PoIFN-gamma in insect cells was confirmed by Western Blot, indirect immunofluorescence assay and indirect ELISA. The antiviral activity assay shows that PoIFN-gamma expressed by the rBac-PoIFN-gamma can efficiently inhibit the replication of the recombinant Vesicular Stomatitis Virus expressing green fluorescence protein in PK-15 cells. The antiviral activity of PoIFN-gamma can be specifically blocked by anti-PoIFN-gamma mouse serum. The antiviral titer of culture supernatant of insect cells infected by rBac-PoIFN-gamma is 2 x 10(4) IU/mL. The results demonstrat that the rBac-PoIFN-gamma can express rPoIFN-gamma efficiently and rPoIFN-gamma has high antiviral activity.
Qin
Li-Ting
LT
Key Laboratory of National Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agriculture Science, Harbin 150001, China.
Wang
Xi-Jun
XJ
Hu
Sen
S
Li
Zhi-Zhong
ZZ
Chen
Wei-Ye
WY
Ge
Jin-Ying
JY
Liu
Si-Dang
SD
Bu
Zhi-Gao
ZG
D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology*; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007106:Immune Sera / Q000276:immunology; D007371:Interferon-gamma / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D011994:Recombinant Proteins; D018411:Spodoptera; D013552:Swine; D018116:Vesiculovirus / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D007106:Immune Sera; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma