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標題: | 建立桿狀病毒作為流感病毒神經胺酸?功能的研究平台 Establishment of Baculovirus as a Platform for the Functional Study of Neuraminidase from Influenza Viruses |
作者: | Yi-Jyun Lin 林怡君 |
指導教授: | 趙裕展(Yu-Chan Chao) |
共同指導教授: | 沈偉強(Wei-Chiang Shen) |
關鍵字: | 桿狀病毒表面表現系統,神經胺酸?,?聯凝集素測定,神經胺酸?抑制性抗體, Baculovirus surface display system,Neuraminidase,Enzyme-linked lectin assay,Neuraminidase inhibition antibodies, |
出版年 : | 2022 |
學位: | 碩士 |
摘要: | 流感 (influenza) 是一種呼吸道疾病,由流感病毒所造成,每年導致全球多達數百萬病例並造成數十萬死亡案件。流感病毒表面具有兩種主要的醣蛋白,血球凝集素 (hemagglutinin;HA)以及神經胺酸? (neuraminidase;NA)。多數情況下,HA佔流感病毒表面80%,針對HA的反應性抗體一直被視為預防流感的主力;然而越來越多研究證據支持,針對 NA 所產生的抗體也對預防流感有重要作用,同時 NA 長久以來也持續被當作抗流感藥物的標的。?聯凝集素測定 (enzyme-linked lectin assay;ELLA) 是一種有效且敏感的方法,可測定神經胺酸?抑制性 (NA inhibition;NI)抗體。在進行 NI 抗體測定時,多會以具刪除血球凝集素的流感病毒作為 NA的來源,以避免血清中會和 HA 結合的抗體干擾 NA 活性,造成偽陽性反應,然而依舊無法完全規避 HA 特異性抗體所導致的誤差。因此本研究利用桿狀病毒表面表現系統,表現 NA 於桿狀病毒表面產生展示 NA 的重組桿狀病毒,嘗試以此重組桿狀病毒作為 ELLA 中替代的 NA 來源,研究是否同樣具有檢測 NI 抗體的效力。我們生成了分別表現來自 A 型流感 11個不同 NA 亞型 (N1-N11),和 2個分別表現來自不同系 (lineage)的B 型流感 NA 的重組病毒,去確認他們是否保有 NA 活性及量化彼此活性差異,結果顯示利用此表現系統表現的 NA 均保有活性。利用電顯可觀察到 NA 成功表現於桿狀病毒表面,IFA,(immunofluorescence assay)結果則顯示,若將此重組病毒感染昆蟲細胞後,NA 會表現在細胞表面。接著再評估 NA-重組桿狀病毒是否可作為 ELLA 中的 NA 來源方面,利用單株抗體和市售可取得的以特定流感抗原免疫過的雞血清,去測定其對於重組病毒的 NA 活性抑制效果,結果顯示單株抗體在抑制 NA 活性上具有極高 NA亞型專一性和敏感性,而雞血清也對相對應 NA 亞型具有較高抑制能力,顯示利用桿狀病毒表面展示系統表現 NA ,可有效用於 ELLA 測定 NI 抗體。接著試著以此系統去檢測田間的豬、牛、羊血清中的 NI 抗體,均可檢測到有個體具有 N2 抑制性抗體反應。 Influenza is an acute respiratory illness caused by influenza virus that causes up to millions of cases of infection and hundreds of thousands of deaths annually around the world. The influenza virus has two main surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA is usually expressed on the influenza virus surface up to 80% and HA-reactive antibodies are typically considered the major factor mediators of protection from influenza infection. However, recent researches suggest that immunity to NA could protect from influenza infection. NA has also served as an important target for antiviral drugs. The enzyme-linked lectin assay (ELLA) is a robust and sensitive method for measuring NA inhibition (NI) antibodies. Whole virus containing an antigenically-mismatched HA is always used in ELLA to avoid non-specific inhibition of NA activity caused by HA-specific antibodies. But HA-reactive antibodies still cause complexity and inaccuracy in the determination of NI antibodies. This study is to express NA on the surface of baculovirus using baculovirus surface display system. After generating the recombinant baculovirus expressing NAs (NA-Bac) from 11 different subtypes of influenza virus A (N1-N11) and 2 distinct lineages of influenza B (Yamagata and Victoria), we characterized the NA activity displayed by these recombinant baculoviruses. In the NA activity test, the NA-Bacs retain native activity and the NA proteins were expressed on the surface of virus under the transmission microscopy. The NAs were expressed successfully on the surface of infected insect cells by the NA-Bacs in the immunofluorescence assay (IFA). The feasibility of NA-Bacs as alternative NA sources in ELLA was also determined. The monoclonal antibodies and commercially available chicken serum that immunized with specific influenza antigens were obtained and used to inhibit the NA activity of NA-Bacs. The results showed that the monoclonal antibody had high NA-subtype specificity and sensitivity against particular NA-Bacs. The chicken serum also showed inhibition capacity of the corresponding NA subtype. It indicates that the NA-Bacs generated by baculovirus surface display system can be effectively used in ELLA to measure NI antibodies. Then this system was also used to detect NI antibodies in the sera of pigs, cattle and goats sampled from the field, showing N2 inhibitory antibody responses from these animals. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/83768 |
DOI: | 10.6342/NTU202202096 |
全文授權: | 未授權 |
顯示於系所單位: | 植物病理與微生物學系 |
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