Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66387
標題: 假性狂犬病毒早期調節基因UL54之選殖與表現及功能分析
Cloning, Expression and Functional Analysis of the Pseudorabies Virus Early Regulatory Gene UL54
作者: 吳金英
Wu, Ching-Ying
關鍵字: Pseudorabies virus
假性狂犬病毒
UL54 gene
early gene
Western blotting
immunoperoxidase staining
UL54基因
早期基因
西方轉漬
免疫過氧化氫酶
染色
出版社: 獸醫微生物學研究所
摘要: 假性狂犬病毒(Pseudorabies virus, PRV)是一種Alphaherpesvirus,其基因結構及生物特性和同亞科的人類單純疱疹病毒第一型(HSV-1)相似。PRV之UL54基因產物屬於早期調節蛋白,與HSV-1的立即早期蛋白ICP27 (infected-cell protein 27)有41%的同源性。由於目前PRV UL54基因相關研究缺乏,故根據兩者間的相似性,針對UL54基因進行選殖與表現,以進一步研究其基因表現產物的功能特性。本實驗以本土株TNL病毒為模板,經過PCR增幅及TA cloning,將UL54基因選殖於pCRII-TOPO載體後,構築出pTOPOUL54質體。將此質體進行定序後,顯示TNL株全長UL54基因coding region為1092 bp,可轉譯出363個胺基酸,此序列已在GenBank登錄,序號為AY101596;與Ka株 UL54基因序列比較,具有97%的相似性。繼而將所選殖的UL54基因轉殖於原核表現系統pET28b後,轉型於大腸桿菌BL21(DE3)表現菌種,以1mM IPTG進行誘導產生重組表現蛋白。經西方轉漬(Western blotting) 分析約在45 kDa處有一明顯誘導蛋白被Anti-His抗體所辨視。進一步純化UL54表現產物,進行小白鼠之免疫以製備特異性免疫血清。經免疫過氧化氫酶染色(immunoperoxidase staining),顯示小白鼠免疫血清確能辨認PRV感染PK15細胞內之UL54病毒蛋白,而且UL54蛋白多分佈於細胞核中。進一步分析UL54蛋白在PRV感染細胞後之表現情形,發現在病毒感染後第四個小時即可偵測到40 kDa之UL54蛋白,且隨著感染時間增加其蛋白量也隨之增加。為了更深入研究UL54蛋白是否具RNA結合功能,我們將UL54基因轉接到pET32b表現載體,構築了一系列UL54之C端缺損的選殖株,經西方轉漬分析証實可分別表現N端33、81、150、261個胺基酸及全長等不同UL54之重組表現蛋白。將各個重組蛋白與Dig標幟之polyG RNA probe進行雜合反應,經Northwestern blotting分析之結果顯示只表現N端33個胺基酸的UL54重組蛋白與pET32b表現蛋白並無任何訊號產生,其餘的UL54重組蛋白與家禽里奧病毒RNA結合蛋白σNS均出現陽性反應,顯示UL54蛋白具有與polyG RNA結合的能力,且在胺基酸序號第34-81序列為與RNA結合所必需。
Pseudorabies virus (PRV) is an alphaherpesvirus, and its gene organization and regulation are similar to the well characterized human simplex virus (HSV). The PRV UL54 gene is an early gene with 41% homology to the essential immediate-early protein ICP27 of HSV. The purpose of this study is to obtain the PRV UL54 gene for further characterizing the gene structure and function. The complete coding region of TNL strain UL54 gene was cloned by PCR cloning strategy, and sequence analysis of this gene revealed that the UL54 consisted of 1092 nucleotides encoding a 40 kDa protein of 363 amino acids. The DNA sequence was submitted to the GenBank data library with the accession number AY101596. The UL54 gene fragment was recovered and subcloned onto the expression vector of the pET28b for producing large scale of UL54 protein in E. coli. system. The expressed UL54 fusion protein was 45 kDa in size which could be recognized by anti-His antibody in Western blotting assay. The expression product was purified and then used as antigen to immunize mice for preparing specific antibodies against UL54 protein. The specificity of the mouse immune serum was confirmed by its ability to react with a 40 kDa viral protein present in the PRV infected cells in Western blotting assay, detected as early as 4 hours after infection. In addition, immunoperoxidase staining of PRV infected cells undertaken with this antibody demonstrated mainly nuclear staining pattern. Furthermore, a series of C-terminal truncated mutants expressing N terminal 33, 81, 150, 261 amino acid residues respectively were constructed for mapping the functional domain of RNA binding. The result of Northwestern blotting was shown that the UL54 protein could bind efficiently to RNA polyG and the N-terminal amino acid residues 34-81 were necessary for its RNA binding activity.
URI: http://hdl.handle.net/11455/66387
Appears in Collections:微生物暨公共衛生學研究所

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