登革病毒(dengue virus; DENV)感染，臨床上會造成嚴重程度不一的症狀，包括輕微的典型登革熱(dengue fever; DF)，甚或演變成嚴重有生命危險的登革出血熱(dengue hemorrhagic fever; DHF)和登革休克症候群(dengue shock syndrome; DSS)，主要病徵有血管滲漏、血小板低下以及出血傾向。至今導致嚴重登革熱感染的發病機制仍未明，於是無法提供專一且有效的治療去預防DHF/DSS。然而，根據先前研究指出，不管是來自DENV感染病患測得自體抗體的血清，抑或是利用DENV非結構性蛋白1(nonstructural protein 1; NS1)多次免疫小鼠的血清中，都存在抗登革病毒非結構性蛋白1之抗體(anti-DENV NS1 antibodies)且能交互作用結合幾種不同的人類自體抗原，包括內皮細胞、血小板以及人類纖維蛋白原，該自體免疫的現象有可能成為解釋嚴重出血熱的免疫致病機制。為了深入研究anti-DENV NS1抗體在出血症狀所扮演的病理角色以及參與的免疫致病機轉，探討宿主對抗病毒感染產生的自體免疫反應是否導致DHF/DSS的發生，更期待研究結果提供臨床診斷或治療途徑更多有用的醫療資訊。首先製備DENV第二型的NS1，重組病毒蛋白以pET-43.1(+)載體構築在大腸桿菌Rosetta菌株表現，產出C端帶有六個組胺酸標記的融合蛋白，接著以鎳離子金屬親和力純化得到的重組蛋白(rNS1)去多次免疫小鼠，得到anti-DENV rNS1抗體的免疫血清，同時發展噬菌體表現系統，利用pComb3X 噬菌粒載體製作出單鏈抗體(single-chain variable fragments; scFvs) 含有抗原決定區(complementarity determining regions; CDRs)的輕鏈和重鏈之變異區。在酵素免疫分析法實驗中，anti-DENV rNS1免疫血清中的多株抗體，能結合重組蛋白、病毒感染自然產生的NS1以及交叉反應於人類纖維蛋白原。此外從免疫rNS1之小鼠脾臟，以PCR方式取得抗體基因變異區部分，與噬菌體外套膜蛋白基因連接表現，各個噬菌體pIII蛋白都可表現一個特定的抗體，經過親和力循環篩選後，挑選出可與抗原rNS1結合且同樣也交互作用於人類纖維蛋白原的專一性結合單鏈抗體，並且透過脫氧核糖核酸(DNA)序列推導成胺基酸序列，進行胚系(germ-line)基因分析找出所隸屬的小鼠抗體家族，也利用終止密碼子(amber codon)的調控，表現純化出重組單鏈抗體片段。該挑選得到的anti-DENV rNS1單鏈抗體，不僅能夠辨認結合人類纖維蛋白原，而且還會導致凝血酶時間的延長，顯示anti-DENV rNS1抗體會干擾纖維蛋白原的活化功能，並可能在DHF/DSS導致凝血功能失常進而在誘發出血傾向扮演一定的角色。

Dengue virus (DENV) infection can cause mild dengue fever or severe life-threatening dengue hemorrhagic fever (DHF)/ dengue shock syndrome (DSS) which are characterized by vascular leakage, thrombocytopenia and hemorrhagic manifestations. Since the mechanism to cause DHF/DSS is not fully understood, there is no specific and effective therapy to prevent its lethality. However, previous studies showed that anti-DENV nonstructural protein 1 (NS1) antibodies (Abs) from both dengue patient sera and DENV NS1-immunized mouse sera have been shown to cross-react with several different human antigens such as endothelial cells, platelets and fibrinogen, which may contribute to the immunopathogenesis of DHF/DSS. To further understand the pathogenic role of anti- DENV NS1 Abs to cause hemorrhage and autoimmunity which may be involved in the pathogenesis and progression of DHF/DSS. The medical information gathered in the present study will be helpful to offer better clinical diagnosis, treatment and vaccine development against DENV infection. Recombinant DENV type 2 NS1 (rNS1) was cloned in E. coli Rosetta using the expression vector pET-43.1a (+) as a C-terminal His6 fusion protein. Purified rNS1 was used to immunize mice for the production of anti-DENV rNS1 mouse sera and the development of monoclonal phage displayed single-chain variable fragments (scFvs) which contain complementarity determining regions (CDRs) using pComb3X phagemid vector system. In enzyme-linked immunosorbent assay (ELISA), we found anti-DENV rNS1 mouse sera bound to rNS1, native form NS1 expressed in DENV-infected cells and human fibrinogen. In addition, anti-rNS1 scFvs which also cross-react with fibrinogen was selected from the scFv library generated from the spleen of rNS1-immunized mice, PCR amplification of antibody variable regions and rapid isolation of specific scFvs displayed on the phage coat protein (pIII) by binding activity in vitro called panning were performed. The DNA fragment of fibrinogen cross-reactive anti-rNS1 scFv clone was sequenced to define the germline gene family. Fibrinogen cross-reactive anti-rNS1 scFv proteins were purified in non-suppressor E. coli strain as soluble form as well as scFv-pIII fusion in suppressor strains. The purified anti-rNS1 scFv proteins not only recognized human fibrinogen but also caused the prolongation of thrombin time (TT), indicating anti-rNS1 Abs can interfere with fibrinogen activation. Taken together, these results suggest anti-rNS1 Abs may contribute to the induction of hemorrhage and the dysfunction of coagulation during DHF/DSS.

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