| Summary: | 碩士 === 國立成功大學 === 醫學檢驗生物技術學系碩博士班 === 98 === 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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