Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24050
標題: 雞貧血病毒結構蛋白VP1於大腸桿菌中的表現、純化及特性之研究
Expression, Purification and Characterization of Chicken Anemia Vrisu (CAV) Structural Protein VP1 in Escherichia coli
作者: 賴冠樺
Lai, Guan-Hua
關鍵字: Chicken anemia virus (CAV);雞貧血病毒;structural protein VP1;expression and purification;結構蛋白VP1;表現與純化
出版社: 生物化學研究所
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摘要: 
雞貧血病毒 (Chicken anemia virus, CAV) 是環狀病毒科唯一的Gyrovirus屬成員。CAV會造成雞隻貧血、淋巴組織萎縮和免疫缺陷,為一免疫抑制病毒。CAV的genomic DNA為一單股環狀DNA,會轉錄出一段帶有部份重疊閱讀框架的mRNA,並且轉譯出三種蛋白分別為VP1、VP2及VP3。VP1蛋白是雞傳染性貧血病病毒的唯一外殼蛋白。由於VP1蛋白對於CAV的生活史扮演相當重要的角色,因此若能闡明VP1的生化特性,相信對於CAV的防治將有極大的助益。為了取得大量VP1蛋白做為研究材料,我們將VP1基因構築於不同的表現載體,並於不同的大腸桿菌中表現重組VP1蛋白,以建立最適VP1蛋白表現的系統及參數。研究結果發現,VP1的表現需要融合蛋白GST的參與,同時藉由最適化VP1蛋白之 N端序列中的rare codon,可於大腸桿菌BL21 (DE3)-pLysS中,明顯增加該重組蛋白的表現量。再者,經過親和性管柱層析純化後的VP1蛋白,於ELISA檢測中可被CAV陽性的雞血清所辨識,顯示該重組VP1蛋白具有極佳的抗原性,可做為開發CAV檢測套組所需的抗原材料。本研究也融合一段綠色螢光蛋白於VP1蛋白C端,轉染進入COS-7細胞後,證實VP1蛋白本身為一入核蛋白 (Nuclear localization protein),具有進入細胞核特性。

Chicken anemia virus (CAV) is the only member of the genus Gyrovirus of the Circovirus. CAV has been proved that is an immune suppressive virus. This virus can cause chicken anemia, lymph organ atrophy and immunodeficiency. CAV genome is a single-strand, circularized DNA, which transcribes a mRNA with partialoverlapped open-reading frames encoded three proteins : VP1、VP2 and VP3。VP1 protein is the only structural protein of CAV and plays an important role in CAV life cycle. It will be useful for CAV prevention and treatment to investigate the biochemical characterization of VP1 protein. To gain more VP1 protein for protein characterization of diagnostic kit development, the VP1 gene was cloned into different expression vectors and then respectively to be expressed using the different Escherichia coli (E. coli) host as expression host. In this study, the expression of CAV VP1 inE. coli was showed that the expression level was significantly increased when the VP1 was fused GST protein. Additionally, by optimizing the rare codons of amino acid residues spanned on the N terminus of VP1 protein, the expression level of VP1 protein in E. coli BL21(DE3)-pLysSwas shown increased obviously. After purification by GST affinity chromatography, the purified GST-Opt-VP1 protein was used to react with CAV-infected chicken serum in an ELISA assay. The results showed that recombinant VP1 protein has good antigenicity to be recognized by CAV-positive chicken serum. This indicated that the purified GST-Opt-VP1 protein is a potential candidate to be an antigen for developing an ELISA to detect anti-CAV antibodies.Finally,the putative nuclear localization signal within the N-terminus of VP1 was also characterized through detection of subcellular distribution of green fluorescent illuminated from VP1 protein fusing GFP, when construction containing VP1-GFP was transfected into COS-7 cells.
URI: http://hdl.handle.net/11455/24050
其他識別: U0005-1808201016215100
Appears in Collections:生物化學研究所

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