Expression of recombinant subgroup A avian leukosis virus gp85 and its application as a diagnostic antigen for antibody detection

家禽白血病病毒（avian leukosis virus, ALV）為雞隻感染引起腫瘤性疾病之病原，在現場因使雞隻產能下降或淘汰增加造成經濟損失。目前市面上家禽白血病A亞群（subgroup A avian leukosis virus, ALV-A）使用全病毒塗鍍作為血清抗體之檢測方式，易因其他亞群抗體而造成檢測之敏感性與特異性不佳。本研究之目的為利用桿狀病毒/昆蟲細胞表現系統進行ALV-A之表面醣蛋白gp85真核表現，並評估此真核表現重組蛋白質應用於免疫酵素連結吸附法之發展潛力。以台灣分離株TW-3577做為表現模板進行gp85基因之表現，建構帶有全段gp85基因的rgp85-baculovirus和全段gp85基因與前導序列之rgp85sp-baculovirus重組桿狀病毒進行Sf9昆蟲細胞之感染。收取重組桿狀病毒感染後之昆蟲細胞，進行西方墨點法分析可偵測到符合預期大小約75 kDa位置之重組gp85蛋白。重組桿狀病毒感染之昆蟲細胞使用間接型免疫螢光染色法也同樣可偵測到重組gp85蛋白之表現。感染72小時之昆蟲細胞cell pellet經lysis後直接做為塗鍍抗原，與標準陽性和陰性血清進行ELISA初測試，確認ALV-A抗血清可造成ELISA讀值之明顯上升，SPF雞隻血清則否。最佳化之ELISA條件使用72個田間樣本血清進行測試，以血清中和試驗做為抗體檢測金標準，得到ELISA之敏感性為80％，特異性為83.3％。利用桿狀病毒/昆蟲細胞表現系統進行之ALV-A gp85蛋白表現，由於重組蛋白無法純化，ELISA之敏感性和特異性略顯不佳，若能成功進行大量蛋白質表現與純化，將可能增加其發展潛力。Avian leukosis viruses, ALVs are the pathogens of neoplastic diseases in chickens, the reproductive reduction and increased elimination may cause the economic loss of chicken flocks. The commercial available subgroup A avian leukosis virus (ALV-A) antibody (Ab) detection kit coated with whole virus may decrease the sensitivity and specificity because of the interfering of Ab from other subgroups. The purpose of the study is using baculovirus/ insect cells expression system to express ALV-A surface glycoprotein (gp85), and to evaluate the potential of the recombinant protein to be used as the antigen in enzyme linked immunosorbent assay (ELISA). Using the laboratory isolate TW-3577 as the cloning template, two recombinant baculoviruses, rgp85-baculovrius and rgp85sp-baculovirus were constructed and infect the Sf9 insect cells. The infected insect cells showed positive result of recombinant gp85 proteins in Western blotting, the target protein size were about 75 kDa. In the indirect immunofluorescence assay, recombinant baculovirus infected insect cell showed positive results reacted with anti-His mAb and ALV-A antiserum. The cell pellet which harvested at 72 hours post infection was lysed, sonicated, and directly used as the coated antigen. 72 field sera were collected to test the optimized ELISA reaction. Using virus neutralization test as the gold standard, the sensitivity and the sepecificity of the rgp85sp ELISA were 80% and 83.3%, respectively. Both values were not good enough for future test due to the impurity of the expressed protein. The purity of the expression protein should be enhanced for future development of this test