Expression of porcine gamma-interferon in recombinant baculovirus and determination of its antiviral activity.
- Author:
Li-Ting QIN
1
;
Xi-Jun WANG
;
Sen HU
;
Zhi-Zhong LI
;
Wei-Ye CHEN
;
Jin-Ying GE
;
Si-Dang LIU
;
Zhi-Gao BU
Author Information
1. Key Laboratory of National Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agriculture Science, Harbin 150001, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Antiviral Agents;
metabolism;
pharmacology;
Baculoviridae;
genetics;
Blotting, Western;
Cell Line;
Enzyme-Linked Immunosorbent Assay;
Fluorescent Antibody Technique, Indirect;
Gene Expression;
Green Fluorescent Proteins;
genetics;
metabolism;
Immune Sera;
immunology;
Interferon-gamma;
genetics;
immunology;
metabolism;
pharmacology;
Mice;
Mice, Inbred BALB C;
Microscopy, Fluorescence;
Recombinant Proteins;
Spodoptera;
Swine;
Vesiculovirus;
genetics;
Virus Replication;
drug effects
- From:
Chinese Journal of Biotechnology
2007;23(3):386-391
- CountryChina
- Language:Chinese
-
Abstract:
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.
