Construction and Growth Properties of Recombinant Pseudorabies Virus Expressing Modified Enhanced Green Fluorescent Protein

( Laboratory of
Animal Virology, College of Animal Science and Veterinary Medicine, Huazhong
Agricultural University, Wuhan 430070, China )

Abstract

The
1.0 kb DNA fragment containing the modified enhanced green fluorescent protein
CDNA M1 (EGFP S147/P) and SV40 poly(A) signal seque nce 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, desig n ated as gG^–/LacZ⁺, was
isolated and confirmed by plaque purification, PCR, So uthern blot and Western
blot. PK-15 cells were infected with the purified recom binant virus at 0.1
pfu/cell and fluorescence emission was monitored at differen t times post-infection
(p.i.) using fluorescence microscopy. Fluorescence emiss ion could be detected
as early as 6 h p.i. when there was no apparent cytopathic effects (CPE) yet.
Fluorescence intensity increased drastically later. Maximu m 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 abov e 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.