Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36058
標題: 傳染性華氏囊病病毒之結構蛋白VP3的表現,純化及特性
Expression, Purification and Characterization of Struture Protein VP3 of Infectious Bursal Disease Virus
作者: 鄭逸新
Cheng, YiHsin
關鍵字: Infectious Bursal Disease Virus;傳染性華氏囊病病毒;nucleoprotein;oligomerization;核酸蛋白;寡聚合作用
出版社: 生物科技學研究所
摘要: 
中文摘要
傳染性雞華氏囊病病毒P3009毒株基因體片段Segment A中較大的開放讀碼區(open reading frame, ORF),能夠指揮轉譯出一多蛋白(polyprotein),經過蛋白酶(VP4)切割成三種蛋白(pVP2,VP4, VP3),其中VP3為一個結構蛋白,其胺基酸序列含KR-rich之特性,並且在C端有一酸性尾端(acidic tail)。因此本實驗將VP3之cDNA序列嵌入表現載體pET28並以大腸桿菌BL21(DE3)plysS進行表現,所表現的VP3為一融合蛋白,其N端含有His-tag(6個histidine)與一段thrombin切位的胜肽。因文獻曾預測VP4-VP3蛋白有兩個不同之截切位置,所以表現了不同N端之HVP3、TVP3蛋白,經His-Bind Resin(Ni-NTA)純化後,所預期的分子量與傳染性雞華氏囊病病毒感染細胞後合成的VP3非常地接近。同時為探討C端在核酸結合所扮演之角色,我們亦表現截切C端一段具核酸結合特性之序列的蛋白(ΔHVP3,ΔTVP3),因此總共在大腸桿菌表現四種蛋白分別為HVP3,TVP3,ΔHVP3,及ΔTVP3。
這四種蛋白與傳染性雞華氏囊病病毒感染細胞後合成的VP3均能被抗IBDV VP3的血清專一的辨識,代表所表現之蛋白確實為VP3融合蛋白。表現之蛋白,以poly(rI)-poly(rC)-Agarose測試其結合雙股RNA之能力,結果顯示:此四種蛋白皆具有此活性。此四種蛋白與32P標定之雙股DNA進行結合反應,結果發現HVP3與TVP3很明顯的會結合dsDNA,而截切C端的ΔHVP3、ΔTVP3蛋白則不具結合能力,代表C端區域可能為一dsDNA結合區。另外一般核酸蛋白所有之寡聚合作用(Oligomerization)的現象,也可在HVP3蛋白與TVP3蛋白和ΔTVP3蛋白中發現。

Abstract
The structural protein, VP3, of the Infectious Bursal Disease Virus(IBDV)contain a highly basic region near its C-terminus(amino acid residues 973 to 998).This region shares sequence homology with several DNA-binding proteins and is presumed to interact with its genomic RNA by stabilizing the negative charge of RNA molecule.
In this work, four recombinant VP3 proteins were expressed in E. coli system using the expression vector pET28. HVP3 protein contained 290 amino acids with six extra Histidine residues and a short peptide of thrombin-cutting sequence at the N-terminus. ΔHVP3, a mutant protein of HVP3, whose highly basic region near its C-terminus was deleted. TVP3, having a different N-end from HVP3, and its C-terminal truncated form- ΔTVP3 were also expressed using the same system. These proteins were purified using immobilized metal-ion affinity chromatography(IMAC)and their soluble forms including refolded ones from inclusion body were tested for their nucleotide-binding activity. All proteins are able to bind poly(rI)-poly(rC)-Agarose, suggesting that VP3 protein has dsRNA-binding activity. Gel mobility shift assay demonstrated that both HVP3 and TVP3 retarded dsDNA in gel but ΔHVP3, and ΔTVP3 have lost their DNA-binding ability. In summary, VP3 protein plays a role of dsRNA binding in IBDV and the highly basic region near its C-terminus is crucial for dsDNA-binding activity.
URI: http://hdl.handle.net/11455/36058
Appears in Collections:生物科技學研究所

Show full item record
 
TAIR Related Article

Google ScholarTM

Check


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