[High level expression of recombinant chicken interferon-gamma in insect cells].
The recombinant transfer vector pFastBacl-ChIFN-y was constructed by plasmid pcDNA-ChIFN-gamma digested with EcoR I and Not I enzymes and cloned into pFastbacl. Then the transfer vector was transformed into E. coli competent cells DH10Bac which contained the bacmid with amini-attTn7 target site and the helper plasmid. The recombinant bacmid-ChIFN-gamma was generated by transposing themini-Tn7 element located in pFastBacl-ChIFN-gamma to themini-attTn7 attachment site on the Bacmid. Subsequently the recombinant Bacmid-ChIFN-gamma was transfected into the Sf9 insect cells mediated by lipofectin to produce recombinant baculovirus and express recombinant ChIFN-gamma (rChIFN-gamma) products. The result showed that the rChIFN-gamma was successfully expressed in Sf9 cells infected with the recombinant virus by indirect immunofluorescence assay (IFA) at 5 days post-transfection. The biological activity of rChIFN-gamma was identified by its inhibition to Vesicular stomatitis virus-induced cytotoxicity of chicken embryonic fibroblasts (CEF) in vitro. The results showed that the most efficient expression of rChIFN-gamma could be obtained at 96h post-infection with multiplicity of infection (MOI) equal to 1. It is interesting that the viruses such as Avian influenza virus H5N1 or Marek's disease virus (GA strain) could not grow in CEF pre-treated with rChIFN-gamma. Cell pathogenic efficient (CPE) in the CEF infected with H5N1 and GA strain is apparently inhibited by the rChIFN-gamma. However only difference between the HA titres of the supernatant of the pre-treated cells is observed without any obvious inhibition effect in CEF infected with Newcastle disease virus (F48E8 strain).
Kong
Gui-mei
GM
Key Laboratory of Jiangsu Preventive Veterinary Medicine, Yangzhou University, China.
Xu
Jin-jun
JJ
Qin
Ai-jian
AJ
Jin
Wen-jie
WJ
Liu
Yue-long
YL
D000818:Animals; D002645:Chickens; D053124:Influenza A Virus, H5N1 Subtype / Q000187:drug effects; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D025922:Mardivirus / Q000187:drug effects; D009522:Newcastle disease virus / Q000187:drug effects; D011994:Recombinant Proteins; D018411:Spodoptera / Q000166:cytology / Q000235:genetics
D011994:Recombinant Proteins; D007371:Interferon-gamma