| Abstract: | 嚴重急性呼吸道症候群（ SARS），是由新發現的人類 SARS 冠狀病毒（ SARS-CoV）所引發的，因為 SARS 冠狀病毒感染肺臟上皮細胞導致病變，並且引發免疫反應如趨化血球的前往。因此認為感染會引發細胞內之基因表達的變化，所以可以做為瞭解分析臨床症狀由來之分子依據。本計畫取得了含有 SARS-CoV 核衣蛋白（ Nucleocapsid）的 pcDNA3 載體，並以 Transient transfection 方法轉殖入肺臟 A549 細胞株內產生該病毒蛋白質，同時於不同時間點（ 0、 12、 24、 48 小時）取出 mRNA 做基因晶片交合實驗。由上述結果，我們取得許多因核衣蛋白在細胞內合成，而引發表達改變之基因的名稱，所以可以進一步分析那一些基因表現之變異可能與 SARS 急性症狀有關。另外，我們完成選殖了非結構蛋白（ NSP） 7-13 未知功能的病毒轉譯區（由 13451 到 22072 位置），成為兩大重疊之 DNA 片段（ 5051-bp 及 5087-bp），並也接入 pcDNA3 載體內，將於近期轉殖入肺臟 A-549 細胞株內。同樣的，也會於不同時間點取出細胞 mRNA 做基因晶片交合實驗，以探討這些 NSP 產生時所引發的基因表現型。當此研究完成時，我們預期將會發現許多人類基因在 SARS 冠狀病毒感染時，參與細胞的生理現象，諸如細胞的生長、退化、凋零等及免疫反應，諸如血液介因子或趨化因子的表達與釋放。
The etiological agent of atypical pneumonia, referred to as severe acute respiratory syndrome or SARS, is a novel strain of human corona virus named SARS-CoV. Patients infected with SARS-CoV may present mild symptoms such as fever, dry cough, and others, but progressive respiratory symptoms may also be resulted from extensive exposure to virus, strong immune response to the virus, or others that lead to severe alveolar damage, lung failure, and finally death. Since the infection of SARS-CoV to the targeted cells induces cell damages and elicits immune response including blood cell chemotaxis, host gene expression patterns induced by SARS-CoV can be served as the molecular basis to understand the mechanism of clinical symptoms of the virus. In this study, we transduced viral nucleocapsid protein in human lung A-540 cell line for 0, 12, 20, and 48 hours and profiled gene expression patterns with cDNA microarrays. We therefore detected many genes whose expression levels were alternated, indicating that these genes may be involved in mediating the symptoms of the viral infections. We have also cloned the viral non-structural genes (NSP) genes 7-13 that contains a large ORF located from 13,451 to 22,072 of viral genome. The ORF was split into two overlapped pieces with sizes of 5051-bp and 5087-bp in length, and inserted into the eukaryotic expression vector pcDNA3. In the near future, these plasmids will be transiently tranduced for viral protein synthesis in A-549 cells, followed by profiling the gene expression patterns. From the results, we anticipate to identify many effector genes that regulate cell physiology such as growth, senescence, and/or apoptosis as well as genes for cytokines or chemokines that mediate the immune responses. |
