title: [Expression of green fluorescent protein using an infectious cDNA clone of infectious bronchitis virus].
abstract: An infectious cDNA clone of H120 vaccine strain of infectious bronchitis virus (IBV) was constructed to demonstrate its potential as a gene transfer vector. Primers were designed according to the published genome sequence of H120 strain, and ten cDNA fragments covering the entire genome of H120 strain was amplified by RT-PCR. All the PCR products were ligated into pMD19-T vector and sequenced, and the ORF5a open reading frame in the pMDTM9 plasmid was replaced by an enhanced green fluorescent protein (EGFP) gene. Recombinant plasmids were digested by the restriction enzyme Bsa I, and all the cDNA fragments were recovered. By using appropriate ligation strategy, the genomic cDNA of H120 strain were reconstituted. Then genome RNA was synthesized in vitro by T7 RNA polymerase and transfected into BHK-21 cells. Recombinant virus expressing the green fluorescent protein was rescued and identified by RT-PCR and sequencing. The characteristics of recombinant virus were evaluated by passage in embryonated chicken eggs. This study showed that the 5a ORF is a good candidate for an insertion site of recombinant genes for the development of IBV infectious cDNA clone as a gene transfer vector.
authors.affiliation: Poultry Institute, Chinese Academy of Agricultural Sciences, Yangzhou 225003, China. dragonsheng@163.com
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhou
authors.forename: Meng-jun
authors.initials: MJ
authors.lastname: Tang
authors.forename: Ya-bin
authors.initials: YB
authors.lastname: Dai
authors.forename: Mei
authors.initials: M
authors.lastname: Liu
authors.forename: Bao-hua
authors.initials: BH
authors.lastname: Zhao
authors.forename: Xu
authors.initials: X
authors.lastname: Cheng
authors.forename: Xiao-juan
authors.initials: XJ
authors.lastname: Lu
mesh_terms: D000818:Animals; D002460:Cell Line; D002642:Chick Embryo; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques / Q000295:instrumentation*; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D001351:Infectious bronchitis virus / Q000235:genetics* / Q000502:physiology
chemical_list: D018076:DNA, Complementary; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins