利用禽流感病毒 RNA 聚合酶 PA 和 PB2 之單株抗體探討其細胞感染機制

Abstract

RNA 聚合酶複合體在禽流感病毒複製過程扮演重要角色，由 PA, PB1與 PB2三個次單元所構成，負責轉錄與複製病毒 RNA，關係到禽流感病毒是否能順利複製病毒子代，感染更多宿主細胞，並決定其遺傳訊息是否能完整傳遞。相較於病毒表面封套上較易突變的醣蛋白 HA 與 NA，RNA 聚合酶複合體在各種病毒亞型間較保守，且其功能乃啟動於病毒感染早期，使其成為良好的抗流感藥物標的。 本論文根據本土禽流感病毒株 A/Chicken/Taiwan/2838V/00 (H6N1) 序列，經結構及免疫性預測，設計可能的抗原決定基，合成短鏈胜肽作為抗原，嘗試製備 RNA聚合酶各次單元體 (PA, PB1及 PB2) 之單株抗體。經酵素連結免疫吸附法 (ELISA) 及免疫染色法分析，確認得到具良好專一性的 PA 及 PB2單株抗體，此外亦得到 PB1之傳統抗血清。分析這些抗體特性，以競爭型 ELISA 初步確認 PA 及 PB2單株抗體之抗原決定位，並以免疫染色法測定 PA 及 PB2 之抗體型別。 為了觀察 RNA 聚合酶各次單元體，於受感染細胞中位移過程，我們使用抗體標定受 H6N1 亞型禽流感病毒株 (A/Wild duck/Ilan/2904/1999) 感染的 MDCK 細胞，確認 PA 及 PB2 單株抗體及 PB1 抗體，均可辨識被感染細胞上的原態抗原。此外，利用免疫共沉澱分析，發現 PA 及 PB1 皆可辨認一個約 53 kDa 大小的蛋白質。另一方面，使用免疫染色法定位 RNA 聚合酶各次單元體於二維電泳圖譜位置，結果 PA 單株抗體可於對應分子量及等電點辨認專一蛋白質點。 本論文所產抗體除了能作為專一性的檢測與觀察工具，更可能進一步應用於基礎研究，協助了解流感病毒 RNA 聚合酶的活化機制。

RNA polymerase is a protein complex composed of three subunits including PA, PB1 and PB2, which play essential roles for the replication of avian influenza virus genome. Compared with the glycoproteins on the viral envelope (haemagglutinin and neuraminidase), RNA polymerase complexe shows more conservative in the protein sequences among various virus subtypes. Moreover, the function of this enzyme complex is initiated in the early-stage of viral replication. This makes RNA polymerase an ideal target for the anti-influenza virus therapeutics. In this study, we used synthetic peptides as antigen for the subunits of the avian influenza RNA polymerase complex (PA, PB1 and PB2) monoclonal anibodies preparation. The peptides were determined based on the structural analysis and the immunogenicity prediction from a local avian influenza virus A/Chicken/Taiwan/2838V/00 (H6N1) sequences. The result of enzyme linked immunosorbent assay (ELISA) and Western blot analysis confirmed that the specificity of the monoclonal antibodies against PA and PB2, as well as the conventional antisera against PB1. The epitopes of these two monoclonal antibodies (anti-PA and anti-PB2) were estimated by using competitive ELISA. The isotypes of the antibodies were determined by Western blots. To follow the cellular movement of the RNA polymerase complexes in a living cell, MDCK cells were infected with the H6N1 virus (A/wild duck/Ilan/2904/1999). Then the target proteins were detected by indirect immunofluorescence assays. The results of indirect immunofluorescence assay shows these antibodies can recognize antigen in native form in infected cells. By using immunoprecipitation, a 53 kDa protein was identified as interacted with PA and PB1. On the other hand,the position of PA on the two-dimensional map was allocated by Western blot. The antibodies produced in this work might be used as a specific probe for viral analysis in order to explore the activation mechanism in RNA replication.