應用NTU1臺灣株大鼠小病毒的類病毒顆粒於ELISA血清抗體診斷試劑之建立

Expression of Virus-like Particles of Rat Parvovirus Type NTU1 and Its Application in Serodiagnosis

張詠惠(Yung-Hui Chang) ； 張照勤(Chao-Chin Chang) ； 王明升(Ming-Hseng Wang) ； 鄭穹翔(Chiung-Hsiang Cheng) ； 萬灼華(Cho-Hua Wan)

《臺灣獸醫學雜誌》 39卷1期 (2013/03) Pp. 39-49

摘要

囓齒類濾過性小病毒（rodent parvoviruses）為囓齒類實驗動物中常見的傳染病病原之一。當rodent parvoviruses感染囓齒類實驗動物或研究用細胞株，對實驗結果的影響甚鉅。因此積極監測實驗大鼠的健康情形，選用適當的診斷工具，在第一時間診斷出病原，才可順利控制感染的情形。本研究以桿狀病毒表現系統，生產新發現的NTU1臺灣株大鼠小病毒（rat parvovirus type NTU1, RPV-NTU1）的主要外殼鞘蛋白（major capsid protein, viral protein 2, VP2），經氯化銫離心純化後於電子顯微鏡下觀察到類病毒顆粒（virus-like particles, VLPs）。利用此RPV-NTU1 VLPs作為抗原，開發出偵測RPV抗體之ELISA（RPV VLP ELISA）。利用具有RPV抗體的大鼠血清以及無特定病原（SPF）之大鼠血清，評估RPV VLP ELISA，發現此ELISA的敏感度（sensitivity）為100%（4/4），且特異度（specificity）亦高達100%（53/53）。此診斷試劑具有使用少量血清檢體、不需犧牲動物、且操作便利、不依賴人工主觀判讀等優點，未來可應用於快速、大量篩檢大鼠的RPV感染。

關鍵字

NTU1臺灣株大鼠小病毒 ； 大鼠小病毒類病毒顆粒ELISA ； 桿狀病毒表現系統

並列摘要

Rat parvoviruses are among the most prevalent infectious agents found in contemporary laboratory rat colonies. Despite the limited clinical diseases and histopathology lesions, rat parvoviruses have significant deleterious effects on research, due to the immunomodulatory effects both in vivo and in vitro and the interference with oncologic research. The objective of this study is to develop a serologic assay to diagnose RPV infection in rats. The major capsid viral protein (VP2) gene of RPV-NTU1, the strain newly identified in Taiwan, was cloned and the recombinant RPV-NTU1 VP2 DNA was expressed using a baculovirus expression system. After purifying the recombinant VP2 protein by CsCl-gradient centrifugation, virus-like particles (VLPs) were observed under electron microscopy. The VLP was then applied as the ELISA (RPV VLP ELISA) antigen to detect anti-RPV antibodies in naturally infected rats. The cut-off optical density (OD) value of 0.25 for the RPV VLP ELISA was set at the value of the mean plus two standard deviations (SD) determined by 40 serum rat samples free from rat parvoviruses infections. Sera from rat infected with RPV (n = 4) and sera from specific-pathogen-free (SPF) rat colonies (n = 53) were used to evaluate the ELISA. The RPV VLP ELISA was sensitive (100%; 4/4) and specific (100%; 53/53) in detecting RPV-infected rats. The RPV-NTU1 VP2 protein provides a ready source of easily-produced antigen and the RPV VLP ELISA developed provides an accurate, high-throughput assay for the serodiagnosis of RPV infection in laboratory rats.

並列關鍵字

Rat parvovirus type NTU1 ； Rat parvovirus virus-like ELISA (RPV VLP ELISA) ； baculovirus expression system