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- Expression and Functional Analysis of the Pseudorabies Virus Immediate-early Protein IE180
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題 名 | Expression and Functional Analysis of the Pseudorabies Virus Immediate-early Protein IE180=假性狂犬病毒立即早期蛋白於真核細胞之表現及功能分析 |
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作 者 | 黃千衿; 張菁雯; | 書刊名 | 臺灣畜牧獸醫學會會報 |
卷 期 | 69:1/2 民88.06 |
頁 次 | 頁37-47 |
分類號 | 437.246 |
關鍵詞 | 假性狂犬病毒; 立即早期蛋白; 激活功能; Pseudorabies virus; Immediate-early protein; Transactivation; |
語 文 | 英文(English) |
中文摘要 | 假性狂犬病毒立即早期蛋自於真核細胞之表現及功能分析假 性 狂 犬 病 毒 ( pseudorabies virus: PRV) 的基因依其表現的先後順序可分為立即早期基因 (immediate-early gene:TE gene)、 早期基因 (early gene) 及晚期基因 (late gene) 三 大類。其中立即早期基因在病毒感染細胞後,不需其他病毒蛋白質協助便可立刻開始表現, 產生一分子量約 180 kDa 的立即早期蛋白 (immediate-early protein), 將其簡稱為 IE180。IE180 為一個 transactivator,可活化病毒早期及晚期基因的表現,因此在整個病 毒感染與複製過程中,扮演了相當重要的角色。 為了進一步了解 IE180 的特性,我們遂將 國內假性狂犬病毒臺南分離株 (TNL strain) 的 IE 基因選殖入 pMAMneo 真咳表現載體上 , 構築成 pMIE 重組質體,再以 pMIE 感染 (transfect)BHK 細胞 (baby hamster kidney cell),並利用免疫過氧化氫酵素染色法 (immunoperoxidase staining) 證實 pMIE 可以在 BHK 細胞內表現病毒的立即早期蛋白。 另外再將 pMIE 與含有假性狂犬病毒 DNase 基因啟 動子 (promoter) 及 CAT 酵素基因構成的 pDNase-CAT 報告質體 (reporter plasmid) 共 同感染 (cotransfect) BHK 細胞, 藉由分析 CAT 酵素的活性,證實 IE180 蛋白質可激活 (transactivate)PRV 早期基因之一的 DNase 基因之表現。此外我們也構築了系列 IE 基因 的缺損 (deletion) 選殖株,分別可表現 IE180 之 N 端 1,440、1,189、736 及 629 個 胺基酸或缺損其中的第 132 至 736 之胺基酸。 經感染 BHK 細胞後,以免疫過氧化氫酵素 染色,證實各突變株皆可於 BHK 細胞內表現 IE 蛋白質。 按著將這些不同的 IE 基因缺損 突變株分別與 pDNase-CAT 質體共同感染細胞, 並利用 CAT-ELISA 進行對各表現蛋白質的 活性分析, 結果顯示 PRV 之 IE 蛋白質 N 端從 1 到 1,189 胺基酸為其執行激活功能所 必須。 |
英文摘要 | The immediate-early (IE) gene of pseudorabies virus (PRV) which is expressed immediately upon infection, encodes a phosphorylated protein (immediate- early protein, IE 180) that can transactivate the viral early and late genes, plays an essential role in regulating viral gene expression.To characterize the transactivation function of IE 180, the IE gene of PRV TNL strain was cloned onto the eukaryotic expression vector pMAMneo, and constructed a recombinant expression plasmid designated pMIE. Plasmid pMIE was then transfected into the baby hamster kidney cells (BHK cells), and the expression of IE protein in the cells was detected by immunoperoxidase staining. In addition, transactivation activity of pMIE-expressed IE 180 protein was analyzed by cotransfection with a chloramphenicol acetyl transferase (CAT) reporter plasmid containing the promoter of PRV DNase gene (pDNase-CAT). The CAT assay showed that pMIE-expressed IE 180 protein could significantly increase the CAT expression, suggesting that this expression product was able to transactivate the PRV early gene (DNase gene).To further identify the activation regions of IE 180, we constructed various truncated mutants of IE gene including the gradual deletions of the carboxyl-terminal coding sequence and an in-frame deletion mutant. Cotransfection of these mutants individually with pDNase-CAT followed by CAT-ELISA analysis demonstrated that truncation of 271 carboxyl-terminal amino acid residues from IE 180 still retained the major function of transactivation, but further deletion of the carboxyl- terminal region up to the amino acid residue 736 resulted in loss of the activity, indicating that the amino-terminal amino acid residues confer transcriptional activation. we have successfully expressed functional IE 180 protein by transfecting the pMIE into BHK cells, and showed that the N-terminal amino acid residues 1-1,189 oflE180 are necessary for its transactivation ability. |
本系統中英文摘要資訊取自各篇刊載內容。