Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene
- Author:
Shun-lin, HU
;
Qin, SUN
;
Qu-zhi, WANG
;
Yul-iang, LIU
;
Yan-tao, WU
;
Xiu-fan, LIU
- Publication Type:Journal Article
- Keywords:
Newcastle disease virus (NDV);
Green fluorescent protein (GFP);
Rescue;
Expression
- From:Virologica Sinica
2007;22(1):34-40
- CountryChina
- Language:Chinese
-
Abstract:
Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain,seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI,which contained the full-length cDNA of the NDV ZJI strain.The pNDV/ZJI,with three helper plasmids,pCIneoNP,pCIneoP and pCIneoL,were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase.After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock,an infectious NDV ZJI strain was successfully rescued.Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP.After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells,the recombinant NDV,NDV/ZJIGFP,was rescued.Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection,indicating that the GFP gene was expressed at a relatively high level.NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route.Four days post-infection,strong green fluorescence could be detected in the kidneys and tracheae,indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.
