| Summary: | 碩士 === 國立中興大學 === 獸醫微生物學研究所 === 86 === White spot baculovirus(WSBV) belongs to the genus Baculovirus
of the family Baculoviridae. Currently, this virus is the most
commonly found virus on shrimp farms and causes severe mortality
in Asia and North America. In the past, diagnosis for WSBV was
based on clinical signs and histopathological observation ;
however, these methods such as the smear test, histopathological
observation, or excrement test often result in the misjudgment
in diagnosis. Recently, the advance of molecular biological
techniques has offered another rapid method for diagnosis of
illness related to shrimp disease with higher specificity and
sensitivity. An accurate diagnosis and early prevention for
WSBV should assist tominimize the mortality rate and reduce the
economic losses. In this study, a Sal I restriction fragment
approximately 4.6 kb in size of the WSBV genomic DNA was cloned
and completely sequenced for nucleotide sequence analysis. Two
primer pairs(3 and 4) were designed according to the nucleotide
sequence of this particular DNA fragment and were subjected to
polymerase chain reaction(PCR) experiments. PCR with primer pair
3 followed by a nested PCR with primer pair 4 could amplified a
specific DNA fragment to WSBV genomic DNA approximately 2.3 kb
in size. Furthermor, a DNA oligonucleotide probe specific to
this WSBV genomic DNA fragment was labeled using DIG labeling
system and used to detect the presence of WSBV DNA in the
infected shrimp by dot blot hybridization or Southern blot
hybridization. The results demonstrated that as little as 100 ng
WSBV DNA could be detected by both methods confirmming the
specificity and sensitivity of this probe. Additionally,
development of serological reagents for WSBV is also important
for diagnosis of this disease. Thus, the nucleotide sequence of
this WSBV 4.6 kb Sal I restriction fragment was further analyzed
by the computer GCG program and revealed six open reading frames
: ORF 1 : 1~282, ORF 2 : 291~1493, ORF 3 : 2073~ 1750 (reverse),
ORF 4 : 2360~2617, ORF 5 : 2971~3687, ORF 6: 3894~4284,
respectively. The ORF 2 which encoded 400 amino acids was
further cloned onto the expression vector pET28b(+) for
production large amounts of this protein in E.coli. The
expressed product of the recombinant plasmid pET28b(+)/pMS321B
was a fusion protein with the molecular weight approximately 70
kDa which was identified by SDS-polyacrylamide gel
electrophoresis and western blotting analysis. This expression
product was further purified by His-bind affinity chromatography
and then used as antigen to immunize mice for preparing specific
antibody against WSBV.
|
|---|
