鴿子疱疹病毒膜醣蛋白D表現及應用

鴿子疱疹病毒（PiHV）為馬立克病毒屬，是疱疹病毒科疱疹病毒亞科家族的成員之一，最初於1945年代所提出。受疱疹病毒感染的幼鴿，死亡率極高，會表現出憂鬱、厭食，臨床症狀如上呼吸道炎症、潰瘍及肝和脾壞死，甚至潛伏於神經系統。綜合上述，需開發以ELISA檢測鴿子疱疹病毒之試劑。本研究大量收集有臨床症狀之鴿子組織樣本，透過聚合酶連鎖反應檢測鴿子疱疹病毒DNA，以研究鴿子疱疹病毒在臺灣之流行率。選殖鴿子疱疹病毒醣蛋白基因克隆至pET24a (+)載體上，轉形至大腸桿菌BL21上做蛋白質表現，誘導生產重組鴿子疱疹病毒醣蛋白。另一方面，將鴿子疱疹病毒之醣蛋白構築於重組昆蟲桿狀病毒載體上，並感染SF9細胞生產鴿子疱疹病毒醣蛋白。將此重組蛋白作為次單位疫苗施打於鴿子上。結果顯示，利用SDS-PAGE及西方點墨法分析重組蛋白，大腸桿菌表現系統生產之蛋白於23 kDa目標位置產生抗體特異性結合，而昆蟲桿狀病毒表現系統生產之蛋白則於41 kDa位置發現目標蛋白。於動物試驗部分，使用昆蟲桿狀病毒表現系統生產之蛋白免疫於鴿子。結果顯示，於酵素免疫分析，免役後14天可見免疫組抗體水平約高於控制組4倍，免疫後28天免疫組抗體水平約高於控制組3.4倍。於細胞激素檢測免疫組之IFN-、TLR 3、TLR 4、TLR 5及TGF-2水平皆有顯著差異，Mx1、STAT1及TLR 2水平則無顯著差異。根據以上結果，可以用於進一步的動物用疫苗研究。

關鍵字

鴿子疱疹病毒；檢測試劑；酵素免疫分析法；次單位疫苗

並列摘要

Pigeon herpesvirus (PiHV) belongs to the Mardivirus genus within the subfamily Alphaherpesvirinae and the family Herpesviridae. It has been reported as early as 1945 that it can cause infections that can lead to high mortality rates in young pigeons, with clinical signs presenting as depression, anorexia or conjunctivitis. Specific lesions can also occur as well as upper respiratory tract inflammation, ulceration, hepatic and splenic necrosis, and even latency in neuron. Thus, an ELISA detection method is needed to be developed for PiHV. In this study, tissue samples from pigeons with clinical symptoms were collected and its DNA was extracted for PiHV by polymerase chain reaction (PCR) and investigate the prevalence of pigeon herpesvirus in Taiwan. Glycoprotein D gene of PiHV was cloned to pET24a (+) then transformed to Escherichia. coli BL21 for protein expression. On the other hand, the glycoprotein D of PiHV was constructed on a recombinant insect baculovirus vector, and infected with SF9 cells to produce PiHV glycoprotein D. This recombinant protein will subcutaneously administered to pigeons as a subunit vaccine. The results showed that using SDS-PAGE and Western blot analysis of recombinant protein, the protein produced by the E. coli expression system produced antibody-specific binding at the target site of 23 kDa, while the protein produced by the insect baculovirus expression system was found at the site of 40 kDa target protein. In the animal test, the protein produced by the insect baculovirus expression system will use to immunize pigeons. The data showed that through visible ELISA, the antibody level 14 days after immunization is about 4 times higher than the control, and the antibody level 28 days after immunization is about 3.4 times higher than the control. And, the levels of cytokines IFN-, TLR 3, TLR 4, TLR 5 and TGF-2 all had significant increased, but there were no in the levels of Mx1, STAT1 and TLR 2. Based on the above results, this can be applied in further animal vaccine research.