Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66268
標題: 假性狂犬病毒(TNL株)立即早期基因之選殖與表現
Cloning and Expression of the Pseudorabies Virus (TNL Strain) Immediate-early Gene
作者: 林盈秀
Lin, Ying-shiow
關鍵字: pseudorabies;假性狂犬病毒;immediate-early gene;cloning;expression;立即早期基因;選殖;表現
出版社: 獸醫微生物學研究所
摘要: 
假性狂犬病毒 ( pseudorabies virus; PRV ) 之立即早期基因 (
immediate-early gene; IE gene ) 在病毒感染細胞後,並不需有其他病
毒蛋白的產生即可立即開始進行轉錄 ( transcription ),在整個病毒複
製週期中扮演一啟始的功能,即可激活 ( transactivate ) 其他病毒基
因或感染宿主細胞的基因表現。本實驗即是利用此病毒之國內分離株 -
台南株 ( TNL strain ) 於含 50 *g/ml cycloheximide 培養液之病毒感
染 MDBK 細胞中進行 IE 基因轉錄體之萃取與分析。所純化的病毒 IE
mRNA 以 PRV 限制酵素 ( restriction enzyme ) BamHI 第 8 片段之
DNA 做為探針進行北方雜合試驗之結果,顯示此病毒只產生單一長約 5.6
kb 之 IE 基因轉錄體。為了更進一步研究此基因產物 ( 即 IE 蛋白 )
之結構與功能,遂進行 IE 基因之選殖與表現。將 PRV BamHI 第 8 片段
之 DNA 經由限制酵素 NcoI 及 BamHI 切割後並選殖於表現載體 (
expression vector ) - pET30a(+) 後分別構築 pN 及 pNB 兩個重組表
現質體。其中 pN 包含 IE 基因之 NcoI/ NcoI DNA 片段而 pNB 則含有
NcoI/BamHI DNA 片段,且兩者皆能成功地利用大腸桿菌 ( Escherichia
coli ) 之 pET 表現系統於 1mM IPTG 誘導之下大量表現 IE 蛋白。而
由 SDS 蛋白質電泳與西方轉漬反應之分析結果顯示,pN 之表現產物約為
60 kDa 而 pNB 則為 100 kDa。此外,利用這些純化後的立即早期蛋白表
現產物進行小白鼠免疫所製備之免疫血清,於 dot blotting 分析與西方
轉漬反應試驗中皆能辨識病毒感染細胞內所產生的 180 kDa IE 蛋白,顯
示其抗體專一性。同時,應用這些抗血清進行病毒感染細胞之
Immunoperoxidase staining 則更進一步顯示病毒 IE 蛋白產生後由細胞
質逐漸進入細胞核內之分佈情形。

The immediate-early ( IE ) gene of pseudorabies virus
expresses immediately upon infection and does not require prior
viral proteins synthesis for its expression. The product of IE
gene can transactivate viral early and late genes as well as
other cellular genes. Thus, the PRV IE gene plays an important
role in initiation of specific gene expressions. The purpose of
this study is to characterize the PRV native strain ( TNL ) IE
gene transcript and then to construct useful recombinant
expression vectors for producing a large amount of IE protein.
The virus was first infected to MDBK cells in the presence of 50
ug/ml cycloheximide and then to extract the total mRNA from
infected cells. The result of northern blot hybridization using
the 8th BamHI fragment of PRV genomic DNA as probe was shown
that there was only one IE mRNA produced about 5.6 kb in size.
Furthermore, in order to study the structure and functions of IE
protein, the IE gene was cloned onto pET 30a(+) expression
vector via NcoI and BamHI cloning sites. Two recombinant
expression plasmids were obtained in which pN contains IE gene
NcoI/NcoI fragment after the initiation codon while pNB contains
the rest of the gene ( NcoI/BamHI fragment ). Both pN and pNB
were respectively transformed into E. coli and could
successfully produce large amounts of IE products in cells
during the induction with 1mM IPTG. The expressed IE proteins
for pN and pNB were 60 kDa and 100kDa in size respectively.
These expression products were further purified by His-bind
affinity chromatograhpy and then used as antigens to immunize
mice for preparation of specific antisera. The specificity of
mice immune sera to IE protein was confirmed by their abilities
to react with IE 180 protein in the PRV-infected cells in the
western blot analysis. Finally, the immunoperoxidase staining
experiment performed with these mouse antisera could localize
the IE protein in the PRV-infected cells and also demonstrated
its transportation from cytoplasm to nucleus during the
infection period.
URI: http://hdl.handle.net/11455/66268
Appears in Collections:微生物暨公共衛生學研究所

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