Cloniog and Application of DNA Probes from Eel Herpesvirus on Formosa (EHVF) Genome

• Main Authors: Hu, Ching-Wen, 胡景雯
• Other Authors: Hsiu-Hui Shih
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/66563896352265879144

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.