| Summary: | 碩士 === 國防醫學院 === 微生物及免疫學研究所 === 99 === Dengue virus (DENV) is a member of the mosquito-borne flaviviruses and causes dengue fever or more severe dengue hemorrhagic fever or shock syndrome diseases over the world and caused approximately 25,000 deaths with 25 to 100 million new infections per year. The mosquitoes Aedes aegypti as well as Aedes albopictus are major vectors to transmit dengue virus to humans. Endemic dengue infection occurs periodically in Taiwan with imported cases from other tropic regions followed by outbreaks of abundant endogenous cases in southern Taiwan, where more Aedes aegypti mosquitoes are breeding, while few endogenous infection in other northern region due to absent of Aedes aegypti. In the past study, C6/36 and CCL125 mosquito cell lines infected with DENVs revealed a decreased tyrosine phosphorylation and DNA binding activity of AaSTAT and AegSTAT, respectively. And in our study, we have found that the NS3 of JEV- also a member of the mosquito-borne flaviviruses- is interacted with AaSTAT by immunoprecipitation.
we have constructed the DENV type1~4 N-terminal histidine (6XHis) fusion protein comprising the NS3 full protein (residues 1 to 618) linked via a flexible glycine linker (Gly4-Ser-Gly4) to an NS2B hydrophilic domain (residues 51 to 94) in pRSET expression vector. The proteins were produced in BL21 (DE3) pLys S induction by IPTG and purified by Ni-NTA agarose (Qiagen). To evaluate DENV NS3 inhibiting mosquito STAT activities, TNT (Promega) products of STAT and STAT fusion with Jak1 domain from Aedes aegypti and Aedes albopictus were put together with the purified NS2B51-94-Glycine linker-NS3 protein in vitro for enzyme activity assay. These mixtures would further to test the disturbance of NS3 protein on the DNA probe binding activity of STAT-Jak1 TNT product by EMSA examination.
This experiment might provide more specific molecular events that the DENV counteract host innate immunity in mosquito, and provide an avenue to further about test the anti-dengue drug development on whole NS3 molecule not just on the N-terminal protease part of NS3.
|
|---|
