title: [Construction and growth properties of recombinant pseudorabies virus expressing modified enhanced green fluorescent protein].
abstract: The 1.0 kb DNA fragment containing the modified enhanced green fluorescent protein CDNA M1 (EGFP S147/P) and SV40 poly(A) signal sequence w as amplified and cloned in frame behind the first eight codons of the nonessential glycoprotein G (gG) of pseudorabies virus (PRV) to yield a transfer plasmid. The transfer plasmid was linearized and cotransfected with the genomic DNA of P RV Ea mutant gG(-)/LacZ(+). The resulting recombinant virus expressing M1, designated as gG(-)/LacZ(+), was isolated and confirmed by plaque purification, PCR, Southern blot and Western blot. PK-15 cells were infected with the purified recombinant virus at 0.1 pfu/cell and fluorescence emission was monitored at different times post-infection (p.i.) using fluorescence microscopy. Fluorescence emission could be detected as early as 6 h p.i. when there was no apparent cytopathic effects (CPE) yet. Fluorescence intensity increased drastically later. Maximum intensity was achieved at 24-36 h p.i. and fluorescence was stable. With the progress of the CPE, fluorescence vanished. The growth properties of gG(-)/M1(+) in tissue cultures were further examined and the titer of gG(-)/M1(+) was similar to that of PRV Ea wild strain and the parental strain gG(-)/LacZ(+). The above results indicated that the recombinant virus expressing the modified EGFP can be used as an in vivo marker to monitor replication and spread, as well as t o study the molecular pathogenesis of PRV.
authors.affiliation: Laboratory of Animal Virology, College of Animal Science and Veterinary Medici ne, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Liu-Rong
authors.initials: LR
authors.lastname: Fang
authors.forename: Huan-Chun
authors.initials: HC
authors.lastname: Chen
authors.forename: Shao-Bo
authors.initials: SB
authors.lastname: Xiao
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: Chuan-Shuang
authors.initials: CS
authors.lastname: Niu
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics; D049452:Green Fluorescent Proteins; D011558:Herpesvirus 1, Suid / Q000235:genetics* / Q000254:growth & development; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D014162:Transfection
chemical_list: D004274:DNA, Recombinant; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins