流感病毒核糖核蛋白複合體對於病毒樣顆粒的佐劑作用

Abstract

流感病毒的核糖核蛋白 (RNP)複合體具有轉錄和複製病毒核糖核酸的功能。流感病毒的核醣核酸在5’端不具有帽狀結構，裸露的三磷酸端會做為維甲酸誘導基因-I (RIG-I)的促效劑(agonist)，活化I 型干擾素及其相關基因包括白細胞介素-1β (IL-1β)等細胞因子誘導發炎的產生，具備疫苗佐劑的條件。流感病毒的病毒樣顆粒（VLP）具有很好的免疫原性，在動物實驗中證明能誘導個體產生很好的抗原特異性以及跨越病毒株交叉反應的抗體。為了產出流感病毒核糖核蛋白複合體的VLP (H5N2 VLP-RNP)，首先，我從H5N2病毒萃取病毒的核糖核酸，經過反轉錄後對四個相關的基因(聚合酶鹼性蛋白1, PB1、聚合酶鹼性蛋白2, PB2、聚合酶酸性蛋白, PA以及核蛋白, NP) 進行放大並建立雙啟動子表達的載體，讓每個質體都能夠同時表達正股的信使核糖核酸以及負股的病毒核醣核酸。以標籤(tag)進行即時定量聚合酶連鎖反應(qPCR)，結果顯示，雙啟動子表達質體在單獨轉染下，兩種核糖核酸約呈等量表達，同時以特異性抗體驗證核蛋白表達。進一步將這四個基因分為兩組，建構二個表達質體，同時轉染到生產VLP的細胞株 (由中研院農生中心蕭培文博士提供)，製備出含有核糖核蛋白複合體的流感病毒VLP。未來若能成功將RNP包裹進VLP將可進一步證實RNP作為疫苗佐劑的可能優勢。

Influenza viral RNP complex is able to catalyze viral RNA transcription and replication. Without cap structure, 5' tri-phosphate viral RNA generated by the RNP complex is an agonist of RIG-I that may induce proinflammatory cytokines and chemokines like IFN-I and IL-1β, a character of vaccine adjuvant. Viral-like particles (VLP) have high immunogenicity and have been proven to increase antibody specificity and cross-reactivity against different viral subtypes in animal studies. In order to generate an H5N2 VLP-RNP, I first extracted viral RNA from the H5N2 virus, amplified and generated dual-promoter expression plasmids after reverse transcription of four related genes (PB1, PB2, PA, and NP), enable all four plasmids to express positive-strand mRNA and negative-strand vRNA simultaneously. As the result, mRNA and vRNA expressed equally in tagged-qPCR analysis after single plasmid transfection, and NP protein expression was also verified through specific antibodies. Further, these four genes were cloned in two expression plasmids and were co-transfected to VLP-producer cells (kindly provided by Dr. Pei-Wen Hsiao from SINICA) to generate influenza VLPs that encapsidated RNP complexes. Advancements of being vaccine adjuvant may be further proved when the RNP complex was well encapsidated in VLPs.