I. 與哈威弧菌結合之多胜肽適合體之篩選 II. 河流弧菌中推定的溶血素之純化

Abstract

I. Vibrio harveyi對蝦業養殖及近海魚類來說為重要致病原，其傳統檢測方式十分費時、費力且結果易於誤判，因此本研究利用噬菌體展示方法篩選出可和V. harveyi結合之多胜肽適合體 (aptamer)，以發展另一種快速檢測方式。首先將噬菌體之七胜肽隨機多胜肽庫用於體外篩選過程，經過五次親和性選擇後挑選31個單一多胜肽噬菌體，將其單股DNA純化定序後發現共有四種胺基酸序列，分別為HHPPWLP、HFDWLPY、LPLNNRL和NSTAIMT，最後以酵素連結免疫吸附法及西方墨點法證明四種多胜肽皆會和V. harveyi結合。

II. Vibrio fluvialis為人類及海洋生物之致病菌，其中溶血素被認為是最重要的致病因子。而V. furnissii之親源關係和V. fluvialis很相近，本實驗室於V. furnissii之胞外分泌液中純化出一具溶血性質的蛋白質，因此推測V. fluvialis可能也有此一性質相似之蛋白質。利用疏水性作用管柱層析分離由V. fluvialis所獲得之胞外分泌液後，經SDS-PAGE電泳分析發現在20% Ethylene Glycol的沖提液中有一約59 kDa的蛋白質，另外也發現此沖提液有溶血活性。進一步跑Native-PAGE後貼在含血培養盤上，也發現具有溶血性質。隨後以V. furnissii 中59 kDa溶血素的抗體進行西方墨點法同樣可辨識到一大小約59 kDa的蛋白質帶。因此推論Vibrio fluvialis之胞外分泌液經過管柱層析分離後，可分離出一具溶血活性的蛋白質。

I. Vibrio harveyi is an important causative agent of vibriosis in penaeid aquacultures and estuarine fishes. Up-to-date, the traditionally methodologies of V. harveyi identification have been time-consuming, labor-intensive, and sometimes can show variable reliabilities. Therefore, we are interested in applying phage display technique for screening V. harveyi-binding peptide aptamers for rapidly medical detection. First, a heptapeptide phage library had been subjected to an in vitro selection process for the identification of V. harveyi-binding peptide aptamers. After five times of selection cycle, 31 phages bearing individually single type of peptide aptamers had been picked, and the single strand DNA from the phages had been purified and sequenced. The result of the selection process is four types of polypeptide sequence found out. Finally, using ELISA assay and Western blotting, the four types of polypeptides had been confirmed that all could bind with V. harveyi.

II. Vibrio fluvialis associates with human and marine organism diseases. And “Hemolysin” produced by Vibrio fluvialis has been known to be a most important virulence factor. On the other hand, a 59 kDa protein functioning hemolytic activity had been isolated from V. furnissii in our laboratory. And the relationship between Vibrio furnissii and V. fluvialis is highly close on the genetics classification. Thus, it is hypothesized that V. fluvialis maybe produces a similar hemolytic protein. Through phenyl sepharose chromatography, eluted proteins had been analyzed by SDS-PAGE and a 59 kDa protein within the 20% Ethylene Glycol fraction had been discovered. In addition to above analyses, the Native-PAGE gel of the fraction coated on blood agar plates had also revealed the hemolytic activity. Subsequently, an ~ 59 kDa protein had been monitored by Western bloting. Consequently, the expected 59 kDa hemolytic protein could indeed be isolated through phenyl sepharose chromatography.