行政院國家科學委員會專題研究計畫成果報告:豬繁殖與呼吸道症候群及假性犬病複合疫苗之開發

Porcine reproductive and respiratory
syndrome (PRRS) and Pseudorabies (PR) are two
impor tant infectious diseases of swine. In order
to construct a chimer ic virus, PR virus was used as
a vector to car ry a foreign virus gene fragment
ORF 3 of PRRS. The total fragment of ORF3
gene was amplified by Rever se-Transcr iption
Polymerase Chain Reaction (RT-PCR). The
resulting cDNA was inser ted into prokaryotic
expression vector (pGEX-2T) system. GST-GP3
(gluthione-S-Transferase-glycoprotein5) expressed
fusion proteins having molecular weight about 30
kDa was obtained. The fusion proteins were used
as antigens to prepare monoclonal antibodies
（Mabs）, and a hybr idoma capabling of secreting
the Mabs against the GP3 fusion proteins was
successfully produced. The Mabs against GP3
fusion proteins was used for screening and
confirming PRRS/PR chimer ic virus car rying the
ORF 3 gene expressed proteins. The Mabs can
identify native GP3 proteins of PRRS virus, but
not to neutralize PRRS virus. . Fur thermore, the
RT-PCR products GP3 gene fragments were
cloned into the pGX vector contained the gG gene
fragment of PR virus. Then, the constructed pJ3X
plasmid DNA contained gG gene fragment of PR
virus and GP3 gene fragment of PRRS virus and
DNA of the attenuated PRV-gE--TK- virus were
co-transfected into RK-13 cells. Following
homologous recombination in the cells, a PRRS/PR
chimer ic virus (designated as J3X chimer ic virus)
car r ied GP3 gene fragment in the genome of PR
virus was successfully constructed. GP3
recombinant proteins of PRRS/PR chimer ic virus
were detected by Southern blotting, Western
blotting, Immunofluorescent assay and
Immuno-peroxidase staining. Plaques produced
by J3X chimer ic virus in RK-13 cell lines were
much smaller than those produced by wild-type PR
virus. The chimer ic virus J3X will be fur ther
investigated on its immunogenicity in pigs.