登革病毒 (DV) 分布於熱帶及亞熱帶區域，是一個由蚊子叮咬所傳播的病毒，造成的疾病包括輕微的登革熱 (dengue fever, DF) 及較嚴重的登革出血熱/登革休克症候群 (dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS)。全球約有超過100個國家、將近一半的人口都在登革病毒的分布的區域內。登革病毒顆粒的表面是由90套膜蛋白 (envelope, E) 的雙聚體所組成， E蛋白是登革病毒最初與宿主細胞接觸的位置，並且E蛋白在病毒感染時的受器媒介胞吞作用 (receptor-mediated endocytosis) 中扮演重要角色。每個E蛋白皆含有三個區域 (domain)，domain I是位於整個E蛋白的中央，domain II含有一段高度保留的融合胜肽 (fusion peptide)，負責E蛋白的雙聚體化，domain III則是一個type-specific並與免疫球蛋白很像的區域，所以也被認為最有可能是受器結合以及具有中和能力抗原決定位的位置。然而，由於抗體依賴性增強作用 (antibody-dependent enhancement, ADE) 造成登革病毒會經由Fc receptor加強感染，到目前為止並沒有一個有效的疫苗可以預防，也沒有有效的藥物可以治療。為了避免ADE的問題，我們利用登革病毒以及E蛋白domain III (DEIII) 免疫小鼠並構築單鏈抗體 (single chain fragments of variable region, scFv) 基因庫，並尋找專一性結合至登革病毒並能中和登革病毒結合及感染的抗體。我們利用PEG沉澱以及蔗糖梯度離心的方式取得純化的登革病毒，以及由pET43.1a(+) vector及細菌表現蛋白系統構築並經Ni2+ column 純化所得到的DEIII蛋白免疫小鼠，經過四次的免疫後，我們可以在其血清中偵測到登革抗體的存在，隨著免疫的次數其效價也有明顯上升的情形，並以流式細胞儀偵測得知以登革病毒免疫小鼠所產生的抗體可以較有效的抑止登革病毒和細胞的結合。萃取免疫小鼠脾臟的RNA並反轉成cDNA，利用PCR建立單鏈抗體基因庫，經過數次的生物篩選後挑選出與登革病毒較高親和力的單一單鏈抗體。在我們的研究裡，總共挑選出了三個單鏈抗體，並利用流式細胞儀分析確認其皆有阻止登革病毒結合的能力，其中以抗登革病毒單鏈抗體5-38的效果為最好。

Dengue virus (DV) is an important mosquito-borne pathogen causing dengue fever (DF), dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) in the tropical and sub-tropical region of the world. Nearly 50% of people in over 100 contries are at risk of DV infection. The surface of DV particle is covered with 90 envelope (E) protein homodimers which contacted with host cells first and were essential for viral infection through receptor-mediated endocytosis. Each monomer of E protein consists of three domains. E protein domain I (DEI) is the central domain located at the center of envelope protein. E protein domain II (DEII) is the dimerization domain contains highly conserved fusion peptide. E protein domain III (DEIII) is a type-specific and immunoglobin-like domain which is also considered a receptor binding and neutralization site. Until today, there is no effective vaccine or treatment against DV infection due to antibodies against DV may enhance DV infection through Fc receptor which is also known as antibody-dependent enhancement (ADE) effect. To avoid this ADE effect, we constructed single chain fragments of variable region (scFv) from DV and DEIII-immunized mice to search for DV specific antibody that can neutralize DV binding and infection. DV purified by polyethylene glycol (PEG) precipitation and sucrose gradient as well as recombinant DEIII expressed in E.coli Rosetta using pET43.1a(+) vector and purified by Ni2+ column were used for immunization. After four times of immunization, antibodies against DV and DEIII were found in both DV and DEIII immunized mice and DV-immunized mouse sera has higher blocking ability against DV binding as detected by flow cytometry. RNA from the spleens of DV-immunized mice was extracted and transcribed to cDNA to establish scFv library. After several rounds of bio-panning, single colonies that had higher affinity to DV were picked up. In our research, we picked up three single scFv that had high affinity to DV and confimed its blocking ability against DV binding by flow cytometry. Among these three scFv clones, anti-DV scFv 5-38 has higher blocking ability against DV binding.

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