APV及PBFDV的分子分析與鸚鵡APV酵素免疫分析 套組之開發

Molecular analysis of APV and PBFDV and the development of a sandwich ELISA for the detection of APV infections in psittacine birds

許志明(Chih-Ming Hsu)

指導教授 ： 蔡向榮

共同指導教授 ： 郭應誠

國立臺灣大學/生物資源暨農學院/獸醫學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.00320

摘要

Avian polyomavirus (APV)及Psittacine beak and feather disease virus (PBFDV)感染症，二者皆為鸚鵡類常見病毒性疾病。鳥多瘤病毒感染症 (APV)，又稱為小鸚哥病 (budgerigar fledgling disease; BFD)，首度於1981年被發現，屬於乳多瘤病毒科 (Papovaviridae)之鳥多瘤性病毒 (Avipolyomavirus)，主要引起年幼鸚哥之急性致死性疾病，其死亡率可高達100%。鸚鵡喙羽病毒感染症(PBFDV)，屬於環狀病毒科 (Circoviridae)之環狀病毒 (Circovirus)，可感染約六十種的野生及寵物鸚鵡。本研究使用聚合酶連鎖反應檢測台灣鸚鵡感染APV和PBFDV之情形，調查時間是從2002年至2005年，共檢驗包含22屬品種165件送檢樣本，結果APV檢測陽性率為15.2%，PBFDV檢測陽性率為41.2%，而混合感染陽性率為10.3%。將部份核酸產物經定序後，與基因庫 (GenBank)之序列比對，APV之VP1基因比對結果相似度為97.5-100%，APV另一個T antigen coding region片段核酸序列與基因庫之APV核酸序列比對，則相似度97.6-100%。PBFDV之ORF V1與基因庫核酸序列比對結果相似度為92.2-100%，另一個ORF C1與基因庫之比對結果相似度為83.3-100%。另以PCR與重組DNA技術，選擇APV-VP1之結構蛋白基因，以原核 (E. coli.)系統成功表現出與預期大小相當之重組蛋白(45 kDa)，此蛋白皆可被特異性之his-tag與抗APV抗體所辨識。利用此APV-VP1重組蛋白作為抗原，免疫BALB/c小鼠，並製備出具分泌抗APV-VP1重組蛋白抗體之融合瘤細胞，經由ELISA、Western blot篩選分析確認，共得8株融合瘤細胞的單株抗體。利用D10-6單株抗體製備Sandwich ELISA套組，用於檢測人工感染虎皮鸚鵡羽囊之APV抗原，並以PCR檢測比較其專一性及敏感性。以二種方法檢測之結果，並無統計學上的差異，証實此Sandwich ELISA可以用於感染APV的虎皮鸚鵡之診斷，並可進一步應用於快速免疫分析試紙。

關鍵字

鸚鵡

並列摘要

Avian polyomavirus (APV) infection and psittacine beak and feather disease (PBFD) are the most common viral diseases of psittacine birds. APV was first isolated from budgerigars in the early 1980s. APV belongs to genes Avipolyomavirus of the family Papovaviridae and causes acute fatal disease in young budgerigars with 100% mortality rate. APV infection is also known as budgerigar fledgling disease. PBFDV, categorized into genes Circovirus of the family Circoviridae, affects over 60 species of wild and captive psittacine birds. In this study polymerase chain reaction (PCR) was applied to diagnosis either APV or PBFDV infections in psittacine birds of Taiwan. From 2002 to 2005, the positive rate of APV, PBFDV, and APV/PBFDV infection over 165 cases were 15.2%, 41.2%, and 10.3% respectively. APVs indicated over 97% nucleotide identity in VP1 and T antigen coding regions. PBFDVs had over 92.2% and 83.3% nucleotide identity in ORF V1 and ORF C1 sequences. Another aim of this study is to prepare Sandwich ELISA for detection of APV and to analyze the specificity and sensitivity of Sandwich ELISA. First of all, we applied prokaryotic (E. coli.) system to express the viral structural protein VP1. The expressed VP1 were specifically recognized by his-tag and anti-APV antibodies. The recombinant protein was used as an immunogen to inject BALB/c mice for preparation of hybridomas which produced anti-VP1 antibodies. Eight monoclonal antibodies secreting hybridomas were obtained as determined by ELISA and Western blot. After screening of these 8 monoclonal antibodies, one monoclonal antibody, D10-6, is used to develop the Sandwich ELISA for the detection of APV. No significant difference was formed between PCR and Sandwich ELISA at the sensitivity. The Sandwich ELISA could be applied for diagnosis of APV infected budgerigars, and could further be applied to immunoassay strip for the rapid diagnosis of APV infection in budgerigars.

並列關鍵字

APV,PBFDV

參考文獻

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