建立細胞分泌型禽流感病毒HA1抗原以檢測疫苗效價與疾病診斷

Development of cell-secreting Avian Influenza Virus HA1 antigen for assessment of vaccine efficacy and disease diagnosis

謝定君(Hsieh Ting-Chun)

指導教授 ： 鄭添祿

高雄醫學大學/生命科學院/生物科技學系碩士班/碩士(2016年)

摘要

台灣在2015年爆發H5N8禽流感(avian influenza, AI)，造成經濟損失和使得安全疑慮升高，為了有效避免鳥禽類流行性感冒的發生，檢測疫苗效價與疾病診斷對抗禽流感病毒(avian influenza virus, AIV) 抗體是重要的，近期血清學診斷技術為瓊脂擴散(agar gel immunodiffusion test, AGID)試驗和血凝素抑制測試(hemagglutination inhibition tests, HI)，然而這些方法主要問題是低敏感性和流程繁瑣，為了克服這些問題，我們計畫發展抗原塗鍍盤透過血液來偵測禽流感H5N8病毒的感染，ELISA血清診斷技術的優勢為，操作簡單、成本便宜和敏感度高，而哺乳類生產的蛋白具有完整後轉譯修飾，保留較佳的抗原性，我們利用帶有HA1細胞外域基因的慢病毒，藉由轉導人類胚胎腎細胞株(HEK293)來產生HA1分泌型HEK293/HA1細胞，我們分泌HA1蛋白質在無血清培養液中濃度高達2μg/mL，於優化HA1塗鍍的效率中，我們發現HA1蛋白質未調整(pH7)條件下，表示HA1蛋白質無須純化或任何調整可直接塗鍍在ELISA盤上，我們以免疫後小鼠來模擬含有抗HA抗體的血清，結果顯示HA1抗原塗鍍盤可評估未經樣本前處理的血清中辨識HA抗體效價，成功開發的診斷測試將可被用來偵測禽類血清中禽流感H5N8病毒抗體，而且我們新發展的抗原盤可以提供便宜和有效試驗來診斷鳥禽類流行性感冒病毒，此外這個ELISA偵測方法，顯然適用於其他抗原的應用，能更有效用於評估抗體效價和疾病診斷。

關鍵字

禽流感病毒 ； 血清學監測 ； 抗原塗鍍盤 ； HA1細胞外域基因 ； HA1分泌型HEK293/HA1細胞

並列摘要

In 2015, the outbreak of avian influenza (AI) H5N8 in Taiwan, caused economic losses and raised safety concerns. To effective prevent AI, serological surveillance of antibodies against avian influenza virus (AIV) is important. Currently, the serological diagnostic techniques are the agar gel immunodiffusion test (AGID) and hemagglutination inhibition tests (HI). However, the major problems of these methods are low sensitivity and very laborious. To overcome these problems, we plan to develop antigen coating plate for detection of avian influenza H5N8 virus infection. We used lentivirus carrying hemagglutinin ectodomain HA1 gene to transduced human embryonic kidney (HEK293) and generated the highly HA1-secreted HEK293/HA1 cells. The production efficacy of HA1 produced by HEK293/HA1 was determined by anti-Myc (as detecting antibody) through indirect ELISA, we secreted HA protein concentrations were as high as 2μg/mL in the serum-free culture medium. For optimization the HA1-coating efficacy, we found the HA1 protein under unadjusted (pH7) conditions, indicating the HA1 protein can be directly coated on the ELISA plate without purification or any adjustment. For HA1-ELISA optimization, we used the HA1 protein coated plate to assess the anti-HA antibody titer in the serum of immunized mice. The result shows the HA1 antigen plate can assess the serum anti-HA antibody titer without sample pretreatment. Successful development of a diagnostic test which can be used to detection of avian influenza H5N8 virus antibody in poultry sera. And our newly developed HA1 antigen plate can provide cheap and effective assays for AIV disease diagnosis. Furthermore, this ELISA has been shown to be suitable for adaptation for use with other antigens in a more effective assessment of vaccine efficacy and disease diagnosis.

並列關鍵字

avian influenza virus ； serological diagnostic ； antigen coating plate ； hemagglutinin ectodomain HA1 gene ； HA1-secreted HEK293/HA1 cells

參考文獻

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