嚴重急性呼吸道症候群B細胞抗原決定位之研究及應用

二零零二年十一月，中國南部的廣東省爆發危及生命且不知病原的非典型肺炎。此疾病具有高度的傳染性及死亡率，藉由直接的接觸快速傳播到全世界。類似的病例在中國、台灣、新加坡、越南、加拿大及美國都有發生。二零零三年二月末，此疾病被定名為：嚴重急性呼吸道症候群。研究報告證實，嚴重急性呼吸道症候群是經由一種新的冠狀病毒所引起，稱為：嚴重急性呼吸道症候群冠狀病毒。過去，我們實驗室研究感染性疾病並且建立運用噬菌體顯現法（phage display），從複雜的血清中，找出與疾病有關的抗原決定位。為了找出嚴重急性呼吸道症候群特有的B細胞抗原決定位，運用噬菌體隨機顯現的胜肽庫對病人血清中的IgG進行篩選的工作。篩選SP1病人血清後，挑選了四十九個呈現陽性反應的噬菌體株，這些噬菌體株都能夠專一的和嚴重急性呼吸道症候群病人血清中的IgG所結合。將這四十九個陽性反應的噬菌體株定序之後，主要分成四組相似性的胺基酸序列，其中兩組胺基酸序列可以對應到嚴重急性呼吸道症候群冠狀病毒基因序列。我們也將噬菌體顯現的序列合成胜肽來進行相關的實驗。在結合及競爭性的實驗中證明，嚴重急性呼吸道症候群病人所產生的抗體的確是由嚴重急性呼吸道症候群冠狀病毒所產生的。陽性反應的噬菌體株及藉由抗原決定位胜肽抗原可以和嚴重急性呼吸道症候群病人的血清反應，證實其確實有臨床診斷上的潛力。利用陽性反應的噬菌體株，可以從四位病人的血清得知體內在感染嚴重急性呼吸道症候群之後IgM，IgG和IgA抗體產生的動力學，瞭解體內免疫反應狀況。此外，從SP3及SP4病人的血清分別篩選出二十四個、六十五個能夠專一和嚴重急性呼吸道症候群病人IgG所結合的噬菌體株，SP3所得的二十四個噬菌體株序列顯現四組相似性的胺基酸序列，而SP4所得的六十五個噬菌體株序列則顯現五組相似性的胺基酸序列。將這些胺基酸序列和嚴重急性呼吸道症候群冠狀病毒基因序列比對之後，SP4所得到的兩種胺基酸序列能對應到嚴重急性呼吸道症候群冠狀病毒的Spike蛋白。總而言之，嚴重急性呼吸道症候群的B細胞抗原決定位序列符合此新的冠狀病毒。這指出此新的冠狀病毒的確涉及嚴重急性呼吸道症候群的致病機轉。藉由抗原決定位所發展的血清學檢測，可以運用於實驗室的偵檢，進一步瞭解研究嚴重急性呼吸道症候群的致病機制。確認病毒B細胞抗原的決定位對於瞭解病毒和抗體的反應情形是非常重要的，藉此可以運用於分子生物學及提供發展病毒檢驗專一的血清檢測試劑及疫苗的資訊。

關鍵字

抗原決定位；嚴重急性呼吸道症候群

並列摘要

In November 2002, an outbreak of a life-threatening atypical pneumonia for which no etiological agent could be identified occurred in Guangdong Province, in southern China. The disease is unusual in its high morbidity and mortality rates and SARS seems to transmit through direct contact that lead to fast transmission all over the world. Cases of similar respiratory illness were subsequently reported globally including China, Taiwan, Singapore, Vietnam, Canada and USA. In late February 2003, the new disorder was designed “Severe acute respiratory syndrome (SARS)”. A growing body of evidence has convincingly shown that SARS is caused a novel coronavirus, called SARS-associated coronavirus (SARS-CoV). In past, we studied infectious diseases and set up the phage display technology to identify disease-specific epitopes from complex serum samples. To find SARS disease-specific B-cell epitopes, phage-displayed random peptide libraries were panned on serum IgG antibodies from SARS patients. From SP1 patient, forty-nine immunopositive phage clones that bound specifically to SARS patient serum samples were selected. These phage-borne peptides had four consensus motifs, two corresponding to amino acid sequences reported for SARS-CoV. Synthetic peptide binding and competition assay further confirmed that SARS patients generated antibodies against SARS-CoV. Immunopositive phage clones and epitope-based peptide antigens demonstrated clinical diagnostic potential by reacting with serum samples from SARS patients. Antibody response kinetics were evaluated in four SARS patients, and production of IgM, IgG and IgA were documented as part of the immune response. Furthermore, from SP3 and SP4 patients, twenty-four and sixty-five immunopositive phage clones that bound specifically to SARS patient serum samples were selected. These phage-borne peptides had four consensus motifs from SP3 and no corresponding to amino acid sequences reported for SARS-CoV. However, there were five consensus motifs from SP4 and two corresponding to amino acid sequences reported for Spike protein of SARS-CoV. In conclusion, B-cell epitopes of SARS corresponded to novel coronavirus. This suggests that novel coronavirus might be involved in the pathogenesis of SARS. Our epitope-based serologic test may be useful in laboratory detection of the virus and in further study of the pathogenesis of SARS. Identification of viral B-cell epitopes is important in understanding virus-antibody interactions at a molecular level and provides information for development of virus-specific serologic diagnostic reagents and subunit vaccines.

並列關鍵字

epitopes ； SARS

參考文獻

Update: Severe acute respiratory syndrome--United States, 2003 (2003). MMWR Morb Mortal Wkly Rep. 52, 357-60.

Updated: outbreak of severe acute respiratory syndrome worldwide, 2003 (2003). MMWR Morb Mortal Wkly Rep. 52, 241-6.

Updated: outbreak of severe acute respiratory syndrome worldwide, 2003 (2003). MMWR Morb Mortal Wkly Rep. 52, 269-72.

Severe acute respiratory syndrome (SARS) and coronavirus testing, United States, 2003 (2003). MMWR, Morb Mortal Wkly Rep. 52, 297-302.

Global surveillance for severe acute respiratory syndrome (SARS) (2003). Wkly Epidemiol Rec. 78, 100-5.

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嚴重急性呼吸道症候群B細胞抗原決定位之研究及應用

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