| 摘要: | 雞傳染性貧血病（Chicken infectious anemia；CIA）是由雞傳染性貧血病病毒（Chicken anemia virus；CAV）所引起的雛雞再生障礙性貧血和全身性淋巴組織萎縮的傳染病，為養雞業者最常遭遇到的病毒性疾病之一。雞傳染性貧血病病毒為環病毒科 (Circoviridae)中之Gyrovirus 屬，其基因體為環型單股DNA，基因組之大小約2.3 kb，病毒外殼呈T=1之正二十面體對稱，病毒顆粒粒徑約23-25 nm，其外殼並無外封套 (envelope)包裹。雞是惟一宿主，本病與傳染性華氏囊病病毒、雞馬立克氏病有協同作用，會從而加重病情，使雞群死亡率上升。關於雞傳染性貧血病之防治，尚無有效的防治措施或方法。除重視日常的飼養管理、做好環境消毒及獸醫衛生措施外，並需定期對傳染性華氏囊病和雞馬立克氏病進行免疫接種。回顧以往CAV 的研究文獻，有關病毒的結構、組裝機轉或疫苗的開發研究仍不多，多數集中於VP3 蛋白的致細胞凋亡方面的研究或CAV 的病理機轉之探討。而諸如病毒顆粒的組裝或有關次單位疫苗的開發研究則屬少數。為瞭解VP1 蛋白對於驅動病毒顆粒組裝過程中所扮演的角色，本研究計劃將以 VP1 似病毒粒子作為研究病毒顆粒的材料，擬探討CAV 之VP1 蛋白對於驅動病毒顆粒組裝過程中所扮演的角色。計畫為期三年，第一年－VP1似病毒粒子組裝影響因子之探討。以表現系統所表現得到的重組VP1 蛋白或VP1似病毒粒子，進行影響組裝及去組裝 (Disassembly)因子的分析。分別以不同物、化學因子如溫度、鹽離子強度、金屬離子濃度、金屬螯合劑、核酸分子等，進行VP1 蛋白/VP1似病毒粒子組裝及去組裝試驗，並分析病毒顆粒組裝及去組裝過程中間體(intermediate)的產生。第二年－VP1、VP2、VP3 蛋白及VP1似病毒粒子之蛋白-蛋白與蛋白-核酸交互作用力之特性分析。利用免疫共沉澱法(immunoprecipitation)、親和性管柱層析(affinity chromatography) 結合Pull-down assay 及以重組桿狀病毒共表現VP1、VP2、VP3蛋白，藉由分析VP1似病毒粒子中之組成份，確認重組蛋白間交互作用力之產生。另外，藉由添加特異性或非特異性的不同核酸分子如ssDNA、dsDNA，以便確認核酸分子在蛋白間交互作用力產生之巨分子功能。第三年－VP1蛋白與VP1似病毒粒子對核酸結合之特異性分析。利用定點突變技術，將 VP1蛋白N端的帶正電荷胺基酸突變，以尋找蛋白質中結合核酸分子的重要區域(Domain) 及關鍵胺基酸。同時透過VP1似病毒粒子組裝分析，亦可同時瞭解影響蛋白分子交互作用力的重要關鍵胺基酸。若能成功以VP1 似病毒粒子研究CAV 組裝的過程，將有助於我們瞭解這些結構蛋白之組裝特性對於環狀病毒本身於寄主中釋出或生活史上的重要性，同時未來更可應用VP1 似病毒粒子加速對次單位疫苗的研究與開發。
The chicken anemia virus (CAV) virus is a 25 nm, non-enveloped, icosahedral virus with a single-stranded, circular DNA genome. The proposed classification of CAV is a new family of viruses, designated Circoviridae. CAV occurs worldwide, and the disease has been described in most countries with a developed chicken industry. CAV is not known to infect birds other than chickens. The principal sites of CAV replication are precursor T cells in the cortex of the thymus and hemocytoblasts in the bone marrow. Anemia results from destruction of the latter. Because of the synergism between CAV and other immunosuppressive viruses such as infectious bursal disease virus, Marek』s disease virus control of the latter is also important. There is no specific treatment and no vaccines currently are available for use, although a live vaccine administered in the drinking water is available. Owing to the researches on CAV structure biology, mechanism of capsid assembly and subunit vaccine development are few. There are three major targets in this 3-years project. Using the virus-like particles produced in insect cell and E.coli by expressing CAV viral proteins, sample characterization of virus particles assembly will be conducted and elucidated. First year progress, the key factors such as metal ion, ionic strength, pH, nucleic acid and others viral proteins effects on capsid assembly will be also concerned and characterized in this stage. Second year progress, the protein-protein interaction of three CAV viral protein will be investigated using immunoprecipitation, Ni-NTA pull-down assay and protein co-expression in insect cell. Besides, protein interaction with nucleic acid will also be involved. In final stage, in this project, the nucleic acid binding specificity to VP1 protein will be conducted and elucidated. Using site-direct mutagenesis, the critical amino acid or domain of VP1 protein for nucleic acid binding will be determined. Lastly, if the above research data proved, it is to be effective to characterize mechanism of capsid assembly. Besides, in this project, three investigators are involved with different expertise. Corporation among them complement the experience and knowledge of one another and provide a good chance of success. |
