研究登革病人血清抗體之B細胞抗原決定位

Abstract

登革熱是經由病媒蚊感染人類的重要疾病，在熱帶及亞熱帶地區登革是一個持續擴張的嚴重公衛問題。根據世界衛生組織2000年的估計，全球每年約有五千萬到一億的人口飽受登革病毒的感染，而登革出血熱則高達五十萬病例，即便如此到目前為止尚無任何安全有效的疫苗可以預防此疾病。 辨識病毒蛋白的抗原決定位有助於疾病治療及致病機轉的瞭解，為了研究登革病人血清之B細胞抗原決定位，我們運用噬菌體隨機顯現的胜肽庫對感染登革第二型和第四型的病人血清進行篩選的工作。呈陽性反應的噬菌體株與登革病人血清具專一性結合能力，並且不與正常人的血清結合。將這些經篩選的噬菌體株定序之後，其胺基酸序列可分成三組：第一組的序列主要與登革出血熱病人的血清具較高度的結合力；第二組的胺基酸序列則與登革熱病人的血清具較高度結合力；第三組為對登革熱及登革出血熱病人的血清均具有結合反應。這幾組具相似性的胺基酸序列可分別對應到登革病毒的殼鞘蛋白 (capsid protein)、套膜蛋白 (E protein) 和非結構性蛋白 (nonstructure protein) 的基因序列。其中DP1-1-54噬菌體株與登革出血熱病人的血清具有較高度的相關性，其胺基酸序列可對應到登革病毒的套膜蛋白；DP3-25噬菌體株與登革熱病人的血清具較高的相關性；DP1-2-10及DP2-2-7分別與登革出血熱及登革熱病人血清均具有結合反應，其中DP1-2-10與DP3-25經比對後並未明顯與登革病毒具相同胺基酸序列，而DP2-2-7則對應到病毒的殼鞘蛋白。 我們也將噬菌體顯現的序列製作成融合蛋白來進行相關的實驗。呈陽性反應的噬菌體株及含抗原決定位的重組融合蛋白可以和登革病人的血清反應，證實具有臨床診斷的潛力。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快速偵檢。此外經由分子層面研究登革B細胞抗原決定位有助於探索病毒和抗體之間的反應，並且對登革出血熱致病機轉的研究有極重要意義。

Dengue is an expanding public health problem in the tropics and subtropical area and it’s the most important arboviral disease of humans. An estimated 50-100 million dengue infections and 500,000 DHF cases occur annually (World Health Organisation, 2000), and its effective vaccine still remains elusive. Recognition of viral protein epitopes is required for the development of useful therapies and understanding the pathogenesis of the disease. To investigate disease-specific B-cell epitopes, human serum antibodies from DEN-2 and DEN-4 infected patients were biopanning with phage-displayed random peptide libraries. Immunopositive phage clones were proved to bind specifically to serum samples of dengue patients but not to normal human serum samples. The binding reactivity of the selected phage clones was divided into three different groups. Some epitopes had higher reactivity to DHF serum samples and the other had greater preference to DF. The third group of the selected phage clones recognized both DHF and DF serum samples. These phage-borne peptides had consensus motifs, which corresponded to amino acid sequences of dengue virus, included capsid (C), envelope (E) and nonstructural (NS) proteins. One of these phage clones, DP1-1-54, had higher reactivity to DHF serum samples which corresponded to amino acid sequences of dengue virus E protein; DP3-25 had greater preference to DF; DP1-2-10, DP2-2-7, recognized both DHF and DF. DP1-2-10 and DP3-25 had no corresponded to amino acid sequences reported for dengue virus. DP2-2-7 had corresponded to C protein. To apply phage-displayed epitopes for diagnosis of dengue patients, a recombinant GST-epitope-fusion protein was produced. Immunopositive phage clones and recombinant GST-epitope-fusion protein demonstrated clinical diagnostic potential by reacting with serum from patients with dengue. Our epitope-based serologic test may be useful in laboratory detection of dengue infection. Furthermore, identification of dengue specific epitopes is important in studying the virus-antibody interactions at a molecular level and address the role of antibodies in the pathogenesis of DHF.