| Summary: | 碩士 === 國立臺灣大學 === 動物學系 === 86 === In an attempt to establish DNA probes from Eel herpesvirus in
Formosa (EHVF), we extracted EHVF DNA from purified EHVF. After
digesting DNA by HindIII or PstI, three DNA fragments were
cloned by random and made into probes by use of the DIG-dUTP
labeling kit. Dot blot hybridization was used to detect the
sensitivity and specificity of the probes. Sothern blot
hybridization was used to check the origin of the probes. At the
end of this research, In situ hybridization was used to detect
the early infection of EHVF. By using CsCl gradient
centrifugation, we obtained three white virus bands. Observation
of negatively stained preparations revealed that the first
(high-density) band was mostly composed of enveloped virions
having diameters of 200 nm. The second and third (low-density)
bands were mostly consisted of immature nucleocapsids with
diameters of 120 nm. After digesting EHVF DNA with HindIII and
ligating it on the vector, pUC 19, we cloned a 1,600 bp DNA. The
DNA was labeled with DIG and named H1-1 probe. Using PstI as
restriction enzyme and pUC19 as a vector, we cloned two DNA
fragments. The lengths of the two DNA fragments are 900 bp and
700 bp. They were named P3-6 probe and P1-1 probe after
labelling them with DIG. Dot blot hybridization was used to
detect the sensitivity and specificity. The limit of detection
for these probes were at least 1.6 pg of pure EHVF DNA. These
probes were able to identify TO-2 cells infected with EHVF.
Uninfected cells and cells infected with Herpesvirus cyprini
(CHV) were negative by the same assay. By southern blot hybri-
dization, we confirm that these three probes were cloned from
EHVF DNA. H1-1 probe was used to detect the early infection of
EHVF by in situ hybridization. EHVF DNA can be detected in TO-2
cells after three hours of infection with -EHVF.
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