H7N9流感病毒神經胺酸酶單株抗體之製備及檢測平台開發

2013年中國爆發人類感染H7N9禽流感疫情，經分析此新型流感病毒後發現，其基因片段由數種低病原性病毒 (low-pathogenicity avian influenza, LPAI)，重排 (reassortant) 而來。禽鳥為H7N9之自然保毒者 (reservoir)，染病後並無明顯病徵，疫情監控相對不易。人類主要感染途徑為接觸帶原禽鳥，受感染人類重症率及死亡率都相當高，且台灣曾有4名境外移入病例，故發展快速篩檢平台成為防疫之重要的議題。流感病毒表面之神經胺酸酶 (neuraminidase, NA) 為重要表面抗原，與血球凝集素 (hemagglutinin, HA) 在不同亞型間皆有高度的變異性。同時NA帶有切割唾液酸與半乳糖間醣苷鍵的酵素催化活性，在病毒散播上為最主要的推手，更是目前開發抗流感藥物與作為病毒篩檢之抗原標的。本研究目標在於針對H7N9之NA製備抑制性單株抗體，並期望此單株抗體能投入篩檢平台之開發，協助H7N9疫情監控。本研究嘗試兩種抗原免疫策略，其一利用軟體預測NA序列上，適合之胜肽片段作為抗原 (NA-1、NA-2及NA-3)，其二乃利用桿狀病毒-昆蟲細胞表現系統，表現N9NA球狀頭部區域重組蛋白作為抗原，分別進行小鼠免疫以製備單株抗體。結果顯示，策略一以NA-3胜肽片段製備得可辨認N9NA重組蛋白之單株抗體，而策略二，最終篩得兩株抗NA單株抗體。經抗體分型試驗，確認此三株抗體皆為IgM。進一步由enzyme linked lectin assay (ELLA) 評估抗體對NA切割醣類之抑制能力，結果發現，策略一以NA-3胜肽片段製備之單株抗體 (1B11)，無抑制NA活性之能力，後續將再針對策略二所得之單株抗體進行分析，期望能得到具有抑制NA活性之單株抗體，將有助於了解NA對宿主的致病機轉。未來更期望單株抗體能投入臨床診斷平台之開發，達到疫情監控及防疫之目的。

關鍵字

A型流感病毒；禽流感；神經胺酸酶；單株抗體； H7N9

並列摘要

In March 2013, the first case of human infections caused by avian-origin H7N9 subtype influenza A virus was confirmed in China. H7N9 was reasserted form a variety of low-pathogenicity avian influenza (LPAI) viruses. In contrast to mild symptoms shown on infected birds, the main reservoir of H7N9 virus, human infections resulted in severe respiratory illness and high mortality. In Taiwan, four patients were confirmed as H7N9 infection cases after returning from China. Therefore, it is a critical issue to improve surveillance of influenza and develop quick clinical diagnosis platform for H7N9. The hypervariable surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), of influenza viruses among different subtypes could serve as good targets for developing monoclonal antibody (mAb) against H7N9 virus. In this study, two strategies were utilized for preparation of NA mAb. One is to immunize mice with synthetic peptides (NA-1, NA-2 and NA-3) derived from H7N9 NA, the other is using recombinant N9NA (rN9NA) expressed by baculovirus. Only NA-3 showed relatively high immunogenicity among these peptides, and the antisera derived from NA-3 could recognize rN9NA. After six boosts, mice were sacrificed to prepared hybridoma. One mAb against NA-3 (1B11) and two mAb against rN9NA (2F08 and 3G08) with high specificity were isolated from strategy1 and strategy2, respectively. All the selected mAb are IgM having κ light chain. Analyzed by enzyme-linked lectin assay (ELLA), mAb 1B11 was found not a NA inhibiting antibody. The NA inhibition for the two mAb derived from strategy 2 will be evaluated with ELLA. Once the NA inhibition capacity of the mAb was verified, these mAb will be applied to the development of clinical diagnose platform for H7N9, such as blocking ELISA and antigen-capture ELISA, in the hope of strengthening the surveillance of influenza virus and disease control in Taiwan.

並列關鍵字

influenza A virus, avian influenza ； neuraminidase ； monoclonal antibody ； H7N9

參考文獻

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H7N9流感病毒神經胺酸酶單株抗體之製備及檢測平台開發

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