Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66426
標題: 假性狂犬病毒早期蛋白UL54之功能區分析
Mapping the Functional Domains of Pseudorabies Virus Early Protein UL54
作者: 黃雅如
Huang, Ya-Ju
關鍵字: 假性狂犬病毒
Pseudorabies virus
UL54基因
NLS功能區
RNA結合能力
墨點轉漬雜合
間接螢光抗體染色
UL54 gene
nuclear localization signal (NLS)
RNA-binding activity
dot blotting hybridization
indirect immunofluorescence assay
Green Fluoesence Protein
GFP
出版社: 獸醫微生物學研究所
摘要: 假性狂犬病毒(pseudorabies virus ; PRV)屬於Herpesviridae,引起豬隻嚴重傳染病—假性狂犬病。PRV的基因結構及生物特性與人類單純疱疹病毒第一型(HSV-1)極為類似;且PRV UL54早期基因與HSV-1的立即早期基因ICP27具有同源性。HSV-1之立即早期蛋白ICP27是一種調節蛋白,主要功能為在病毒複製後期能調控早期及晚期基因的表現,也因而抑制宿主細胞的基因表現。ICP27蛋白之結構與功能已被研究較為清楚,其N端胺基酸序列上包含nuclear export signal (NES)、nuclear localization signal (NLS)及RGG box RNA binding domain等三個功能區。本實驗目的為針對PRV UL54蛋白之RNA binding能力及在細胞內之分佈情形進行分析,以進一步決定出NLS及RNA binding功能區之相關位置。首先利用實驗室先前已構築完成含全長UL54基因及一系列C端缺損之重組表現質體,以及另外構築中間部位缺損之重組質體,一同利用大腸桿菌大量表現各種UL54缺損蛋白。表現蛋白經純化後,以dot blotting hybridization方法,進行與poly(G) RNA之結合能力測定,結果顯示N端片段少於83個胺基酸或是中間部位缺損第35-82個胺基酸的重組蛋白則失去與RNA結合之能力。進一步將各種包含特定UL54基因片段之DNA轉殖於真核表現載體pcDNA4/HisMax B,接著將這些真核重組質體分別轉染(transfection)到PK15細胞,進行暫時表現(transient expression)。利用抗UL54蛋白之老鼠免疫血清進行間接螢光抗體染色(indirect immunofluorescence assay),結果顯示含有N端83個胺基酸以上之缺損蛋白皆可在細胞核內出現明顯螢光訊號。並進一步利用可表現UL54蛋白N35及N83之GFP綠色螢光融合蛋白的重組表現質體,經轉染入PK-15細胞進行短暫表現後,結果也顯示N83-GFP融合蛋白主要分布於細胞核中。由以上結果顯示,在UL54蛋白N端83個胺基酸序列為NLS及RNA binding功能所必需,且在病毒基因調控上扮演相當重要之角色。
Pseudorabies virus (PRV) belongs to the family Herpesviridae, and is the causative agent of Aujesky's disease. PRV gene organization and regulation are similar to well characterized human herpes simplex virus (HSV-1), and the PRV UL54 gene is an early gene with homology to the essential immediate-early protein ICP27 of HSV-1. The major function of ICP27 regulatoy protein is to regulate the expression of early and late genes at post- transcription level and inhibit the expression of cellular genes during infection. The N-terminal half of ICP27 has three important function domains: nuclear export signal (NES), nuclear localization signal (NLS) and RGG box RNA binding domain. The purpose of this study was to identify the functional regions conferring for nuclear location and RNA-binding activity. Several recombinant expression plasmids containing various coding regions of UL54 gene were constructed for producing a series of C-terminally truncated or internally deleted forms of UL54 mutants in E. coli or porcine kidney (PK-15) cells. RNA binding activity of E. coli-expressed UL54 mutants was characterized by the binding ability to poly(G) RNA homopolymer. The result of dot blotting hybridization was shown that N-terminal 83 residues of UL54 are responsible for RNA binding and the region of residues 35-82 containing an RGG box is necessary for its function. Furthermore, the region responsible for nuclear localization was investigated by transient expression of various deletion mutants in PK-15 cells followed by detection of their subcellular distribution. The results showed that C-terminal deletion beyond the amino acid residue 83 or internal deletion containing the RGG box sequence could restrict UL54 mutants in the cytoplasm. The ability of the N-terminal 83 residues to target the green fluorescence protein to the nucleus confirmed further its role as a functional nuclear localization signal (NLS). The utmost N-terminal 83 residues portion of UL54 contains two important functional domains, NLS and RNA binding, and thus it wound play an indispensable role in UL54 regulatory function.
URI: http://hdl.handle.net/11455/66426
Appears in Collections:微生物暨公共衛生學研究所

文件中的檔案:

取得全文請前往華藝線上圖書館

Show full item record
 
 
Citations:


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.