- 學位論文
利用桿狀病毒表現系統表現曲弓熱結構蛋白及其p62蛋白質研究
Expression and Analysis of the Structural Protein p62 of Chikungunya Virus by Using Baculovirus Expression Vector System
摘要
中文摘要 曲弓熱病毒 (Chikungunya virus , CHIKV)是透過白線斑蚊進行傳播的病毒，由於有突變株的產生，使得曲弓熱病毒可由亞洲虎蚊(Aedes mosquitoes）帶原傳播，增加了其感染地區，因此如何防治曲弓熱病毒的擴散，以及找出有效的疫苗或者藥物進行治療，是重要公共衛生議題。曲弓熱病毒屬於阿爾法病毒屬，其病毒的結構蛋白的形成，是藉由一條多聚胜肽鏈 (capsid-p62(E2-E3)-6K-E1)進行切割的方式，其形成病毒結構的機制頗具其特色。我們實驗室已成功的使用桿狀病毒表現系統，於昆蟲細胞表現曲弓熱病毒的結構蛋白，並發現其蛋白有使昆蟲細胞融合的能力，進一步的分析顯示引發細胞膜融合現象是由曲弓熱病毒的E1結構蛋白的作用。除了E1結構蛋白，p62結構性蛋白是曲弓熱病毒的另一個重要的結構蛋白，因此本研究對p62(E3-E2)蛋白進行點突變 (site directed mutagenesis)與並分析此些突變p62蛋白對E1蛋白的結合及膜融合現象的研究及分析。透過蔗糖梯度離心實驗以及化學交聯反應，(E1-E2)會形成3聚體形成的結構於重組桿狀病毒之感染昆蟲細胞的細胞質內，而對E2蛋白的C22S，C220S胺基酸進行突變以及弗林蛋白酶 (furin cleavage site; R324A R325A)，觀察到C22S，C220S胺基酸的突變有增強膜融合的現象，弗林蛋白酶切割位的突變則有阻斷膜融合的現象。有文獻報導指出若降低膜融合的現象則可以增加弓熱病毒的類病毒顆粒(virus like particle, VLP)的產生，因此弗林蛋白酶切割位的突變株，將有助於生產曲弓熱病毒的 (VLP) 而發展曲弓熱病毒疫苗生產的潛力。
並列摘要
Abstract Cungunyungunya virus (CHIKV) is originally spreaded through mostiquito Aedes albopictus. However, the generation of mutants make CHIKV can be transmitted by Aedes mosquitoes, therefore result in the expansion of infected areas. How to prevent the spreading of Chikungunya virus and discover the effective vaccines or medicines against CHIKV becomes an important issue for public health. CHIKV belong the genus Alphavirus. The formation of viral structural proteins is accomplished by sequential cleavages of a polypeptide chain (capsid-p62 (E2-E3)-6K-E1), and the mechanism of forming the viral structures of CHIKV has its distinctive features. Our laboratory has successfully expressed the structure protein of CHIKV in insect cells by using baculovirus expression vector system. We observed the syncytial formation in the infected insect cells, and the further analysis showed this phenomenon was resulted from the E1 protein of CHIKV. In addition to the structural protein E1, the p62 (E3-E2) protein is also an important structural protein of CHIKV. In this study, we generated the recombinant viruses with the point mutations on the p62 protein through site-directed mutagenesis and then analyzed the association of these mutant p62 proteins with E1 protein and the influences for the syncytial formations in insect cells. Through the analysis of sucrose gradient density and chemical crosslinking reactions, the heterodimer of E1-E2 proteins could form trimerized complexes in cytoplasm of infected Sf21 cells. When further exammed the infection of Sf21 cells with recombinant viruses which had mutations on E2 protein (C22S and C220S) or furin cleavage site between E3-E2 proteins (R324A/R325A), we found that C22S, C220S mutants might cause an enhanced syncytium formation, but the mutation of furin cleavage site otherwise blocked the cell fusion. According to other related studies, the reduction of the phenomenon of membrane fusion can increase the production of virus-like particles (virus like particle, VLP) of CHIKV. Therefore, we suppose that the mutant virus on the furin cleavage site of p62 protein could have potentials to help produce VLP of CHIKV for the development of CHIKV vaccines.
參考文獻
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