鰻魚皰疹病毒檢驗探針的選殖與應用

Abstract

本研究由純化之鰻魚皰疹病毒（Eel Herpesvirus in Formosa, EHVF）抽出其DNA，經核酸限制?切割後，再以逢機選殖法選殖出三段DNA，將選殖出之DNA以digoxigenin-dUTP (DIG-dUTP)標識後製成檢驗探針。以點墨雜合法測量探針的靈敏度與專一性、南氏雜合法確定探針的來源，最後以原位雜合法將探針應用於病毒初期戚染之檢驗。 EHVF經氯化銫梯度離心後，得到3條白色帶。分別抽出並去除氯化銫成份，用電子顯微鏡負染色檢驗，發現第一條病毒帶的病毒多為成熟並具外套膜（envelope）者，直徑約為200nm；第二、三條病毒帶則多為不成熟且呈二十面體狀之核酸鞘（nucleocapsid），直徑約為120nm。 將EHVF DNA用HindIII限制?切割，接在載體pUC19上，以逢機選殖的方法選殖出一段長為1,600bp的DNA片段，經DIG標識後製成H1-1探針；以PstI限制?切割EHVF DNA，同樣以pUC19為載體，經逢機選殖則選殖出二段DNA，長度分別為：900bp及700bp，以DIG標識後製成P3-6探針及P1-1探針。 以點墨雜合法測量探針的靈敏度與專一性發現：探針單獨呈色之靈敏度分別為：1pg（H1-1探針）；67pg（P3-6探針）；6.3pg（P1-1探針）。探針與EHVF DNA雜合反應之靈敏度則皆為1.6pg。三種探針皆會與由EHVF感染之TO-2細胞抽出之DNA雜合呈色，但不與未被EHVF感染之TO-2細胞抽出DNA或被鯉魚皰疹病毒（CHV）感染的FHM細胞之DNA雜合呈色，因此探針具專一性。且以南氏雜合法檢驗探針的來源發現：三個探針皆是由EHVF DNA（長度與探針相對應的片段）選殖而來。 應用H1-1探針做原位雜合法時發現：EHVF感染TO-2細胞3小時後就可以在TO-2細胞之細胞核偵測到病毒DNA存在。可見使用此探針可以在病毒感染的初期就檢驗到病毒DNA。

In an attempt to establish DNA probes from Eel herpesvirus in Formosa (EHVF), we extracted EHVF DNA from purified EHVF. After digesting DNA by Hind III or Pst I, 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 Hind III 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 Pst I 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.