https://scholars.lib.ntu.edu.tw/handle/123456789/164223
標題: | 抗禽流感病毒非結構蛋白單源抗體之特性分析 Characterization of Monoclonal Antibodies against Nonstructural Protein 1 (NS1) of Avian Influenza Virus |
作者: | 錢品澄 Chien, Pin-Cheng |
關鍵字: | 禽流感病毒;非結構蛋白;酵素聯結免疫吸附法 | 公開日期: | 2014 | 摘要: | 由於 NS1 為非結構蛋白,只有在病毒複製過程才會產生,是判斷動物是否為流感病毒 (influenza virus, IV) 自然感染的重要指標,而目前尚未有 NS1 Ag檢測套組,故本研究利用小鼠免疫 A/chicken/Taiwan/2838V/00 (H6N1/2838) 來源 E. coli 表現之重組 NS1 蛋白,製備抗 NS1 單源抗體 (αNS1 MAb),以間接免疫螢光抗體染色法 (indirect immunofluorescence assay, IFA)、西方墨點法 (western blot, WB)、免疫組織化學染色法 (immunohistochemistry stain, IHC)、真核表現系統 (eukaryotic expression system, EES)、序列比對 (sequence alignment) 等方式進行特性鑑定與分析,進而開發 MAb-based NS1 Ag sandwich ELISA。本研究中 16 種αNS1 MAb 均已經過 H6N1/2838, A/chicken/Taiwan/1209/03 (H5N2/1209) & A/chicken/Miaoli/2904/00 (H6N1/2904) 來源之IFA、WB 與 IHC 等特性鑑定,在真核表現 NS1-F (full length; residues 1-230) 之 IFA 均為陽性;其中有 4 種αNS1 MAb (4M4, 4M6, 4N5, 4R3) 在 NS1-△F (C-terminal deletion; residues 1-207) 之 IFA 呈現陰性,其餘均為陽性。根據 epitope mapping 的分析結果,可將 16 種 αNS1 MAb 分成四大群 (group A, B, C & D),並且主要辨識的位置在 effector domain (residues 74-230),其中 group B 辨識之 epitope 為 C-terminal tail (residues 202-230)。以 αNS1 MAb 分別作為 docking Ab 與 tracer Ab,rNS1 作為抗原進行 sandwich ELISA 條件之優化,挑選表現最佳之組合 (MAb pair, MP);結果顯示,合併 MP-5、MP-9 (4R11 – 4J12/HRP) 與 MP10 (4M2 – 4M2/HRP) 可以偵測 15 種不同 IV 亞型接種 CAM lysate 之 NS1 Ag,故我們相信這三個 MP (MP-5, MP-9 & MP-10) 有潛力應用於檢測不同 IV 亞型之 NS1 Ag。 NS1 is an indictor Ag of influenza virus (IV) infection and it’s only produced during IV replication in very early stage of infection. It can be used in determining whether the chicken was infected or not, which also can be applied on early diagnosis of IV infection. Therefore, my study aimed to characterize the anti-NS1 MAb (αNS1 MAb) by indirect ELISA (iELISA), indirect immunofluorescence assay (IFA), western blotting (WB), immunohistochemistry stain (IHC) and mapping the antibody binding site by eukaryotic expression system (EES). Subsequently, to develop a rapid, sensitive and specific diagnostic MAbs-based NS1 Ag sandwich ELISA that might be incorporated with NP and M Ag ELISA kit for detecting the IV infection. The parental hybridoma have been prepared by immunizing Balb/c mice with E. coli expressed recombinant nonstructural protein 1 (rNS1) oringinated from IV isolates of A/chicken/Taiwan/2838V/00 (H6N1/2838). 16 αNS1 MAbs have been characterized by WB, IFA and iELISA with NS1 Ag from several IV isolates such as A/chicken/Miaoli/2904/00 (H6N1/2904), A/chicken/Taiwan/1209/03 (H5N2/1209). Meanwhile, the epitope mapping was studied in EES. Expression of full length rNS1 (residues 1-230) were all positive and 4 αNS1 MAb (4M4, 4M6, 4N5, 4R3) were negative in C-terminal deletion (residues 1-207). Accroding to epitope mapping analysis results, which can divide 16 αNS1 MAb into four groups (A, B, C & D). The predictive epitopes of 16 αNS1 MAb mainly recognize the effector domain (residues 74-230) and group B recognize the C-terminal tail (residues 202-230) of NS1 protein. MAb-based NS1 Ag sandwich ELISA was designed as sixteen MAb individually conjugated with HRP as the tracer Ab paired with each non-conjugated MAb acted as docking Ab respectively to compare the binding ability of those combinations with E. coli expressed rNS1. Current results indicated that MP-5 (4R11 – 4M2/HRP), MP-9 (4R11 – 4J12/HRP) and MP-10 (4M2 – 4M2/HRP) can detect NS1 Ag from 15 subtypes of IV (H1~H15). It is believed the three MP (MP-5, MP-9 & MP-10) we studied have potential to be applied on detecting the IV NS1 Ag. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/264504 | Rights: | 論文公開時間:2019/08/05 論文使用權限:同意有償授權(權利金給回饋學校) |
顯示於: | 獸醫學系 |
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ntu-103-R01629014-1.pdf | 23.32 kB | Adobe PDF | 檢視/開啟 |
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