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Title: | 桿狀病毒表現家禽網狀內皮增生症病毒封套蛋白質之酵素連結免疫吸附法檢測抗體開發 Detection of reticuloendotheliosis antibody by enzyme-linked immunosorbent assay with baculovirus expressed envelope protein |
Authors: | Ting-Hsiang Hsiao 蕭庭翔 |
Advisor: | 王金和(Ching-Ho Wang) |
Keyword: | 家禽網狀內皮增生症病毒,桿狀病毒/昆蟲細胞表現,重組封套蛋白質,間接型酵素連結免疫吸附法 (ELISA), reticuloendotheliosis virus,baculovirus/insect cells expression,recombinant envelope protein,indirect enzyme linked immunosorbent assay (ELISA), |
Publication Year : | 2014 |
Degree: | 碩士 |
Abstract: | 家禽網狀內皮增生症病毒(avian reticuloendotheliosis virus, REV),可感染多種鳥禽類,包括雞、鴨、鵝、雉及孔雀等。被感染之家禽並未出現明顯病徵,故於台灣家禽疾病中較不被重視,但其卻會引起飼養場重大的經濟損失,於現場尚未有有效預防及控制之方法,為此需開發有效檢測REV之方法。本研究之目的為利用桿狀病毒/昆蟲表現系統進行REV封套蛋白質之真核表現,並評估其重組蛋白質應用於抗體之酵素連結免疫吸附法之發展潛力。以台灣病毒株goose/3410/06之封套序列作為模板,以此建構帶有封套基因之baculovirus-env重組桿狀病毒並進一步感染Sf9昆蟲細胞,收取受感染之昆蟲細胞,進行西方點墨法分析可檢測到符合預期大小約62 kDa位置之重組封套蛋白質,並通過鎳離子親和力管柱進行純化。將重組封套蛋白質塗鍍作為抗原,以棋盤格方式獲得間接型ELISA之最佳化條件,並以血清中和試驗作為抗體檢測之金標準,比較182個田間樣本血清,其中種雞138隻及台灣土雞44隻,結果顯示重組封套蛋白質用於間接型ELISA之敏感性為88.5%及特異性為97.7%。其於敏感性及特異性皆有相當不錯之結果,表示若應用於現場初步診斷使用,應具有良好之發展潛力。額外與實驗室先前所研究以大腸桿菌表現重組封套蛋白質之阻斷行ELISA進行比較,以相同樣本進行測試,結果顯示敏感性為84.6%及特異性為95.3%。此兩種方式所檢測結果相似,但本實驗所發展之間接型ELISA具有所需使用樣本量低、二抗取得容易及成本低等優點。 Reticuloendotheliosis virus (REV) infects variety animals including chickens, ducks, geese, pheasants, peafowl and other birds. REV is ignored in Taiwan because the infected poultry without obvious clinical signs. REV causes severe economic losses in commercial poultry, such as runting disease syndrome and immunosuppression. Currently, the disease lacks effective prevention and control in the field. The purpose of this study is to develop a rapid, reliable, convenient and economic method for detecting REV antibody. We use baculovirus/insect cells expression system to express REV envelope protein, and to evaluate the potential of the recombinant protein to be used as the antigen in enzyme linked-immunosorbent assay (ELISA). To use the laboratory isolated Taiwan strain goose/3410/06 as the cloning template. The full-length of REV envelope was cloned into bacmid vector and then the constructed recombinant baculoviruse-env was infected the Sf9 insect cells. The infected insect cells showed expected recombinant envelope protein in western blotting which size was about 62 kDa. The expressed recombinant envelope protein was purified by Ni-NTA (nitrilotriacetic acid) column. The purified recombinant envelope protein used as the coated antigen. Then, we test optimization condition of indirect ELISA by checkerboard method. Using 182 field samples (138 broilers and 44 Taiwan Country Chickens) to compare virus neutralization tested as the gold standard with the indirect ELISA. The result showed the sensitivity and the specificity of the indirect ELISA were 88.5% and 97.7%, respectively. Both values were good enough to screen detection in the field. The indirect ELISA of recombinant envelope protein possesses potential of development. Additional, we used blocking ELISA which envelope protein was expressed by E. coli to compare with the result of indirect ELISA. The result showed the sensitivity and the specificity were 84.6% and 95.3%, respectively. The two kind of methods displayed similar result, but the indirect ELISA possess advantages of lower usage amount of samples, easier obtainable secondary antibody and cheaper. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57202 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 獸醫學系 |
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ntu-103-1.pdf Restricted Access | 3.29 MB | Adobe PDF |
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