Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66277
標題: 假性狂犬病病毒立即早期蛋白於真核細胞之表現及功能分析
Expression and functional analysis of the pseudorabies virus immediate-early protein.
作者: 張菁雯
Chang, Jing-Wen
關鍵字: pseudorabies virus;假性狂犬病病毒;immediate-early protein;eukaryotic expression;立即早期蛋白;真核細胞表現
出版社: 獸醫微生物學研究所
摘要: 
摘要
假性狂犬病病毒(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;IE 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,不須其他病毒蛋白質協助便可立刻開始表現,產生一分子量
約180KDa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病毒臺南分離株(TNL strain)
的IE 基因選殖入pMAMneo真核表現載體上,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(trans-activate)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。接著將這些不同的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。

Abstract
Abstract
Pseudorabies virus (PrV) is an important swine pathogen that
replicates Pseudorabies virus ( PrV ) is an important
swine pathogen that replicates lytically in the periphery and
can establish a latent infection in sensory lytically in
the periphery and can establish a latent infection in sensory
neurons. PrV genes are generally classified into three kinetic
classes, defined neurons. PrV genes are generally classified
into three kinetic classes, as immediaue-early, early, and late,
on the basis of their temporal expression defined as
immediate-early, early, and late, on the basis of their temporal
during the viral lytic cycle. The 180 KDa immediate-early
protein (IE180) of PrV expression during the viral lytic
cycle. The 180 KDa immediate-early protein functions as a strong
transactivator for viral early and late promoters. In (
IE180 ) of PrV functions as a strong transactivator for viral
early and order to characterize the transactivation function of
IE180, the IE gene of PrV late promoters. In order to
characterize the transactivation funTNL strain was cloned into
the eukaryotic expression vector pMAMneo, and
constructed a recombinant expression plasmid designated pMIE.
pMIE was then transfected into the baby hamster
kidney cells (BHK cells), and expression of IE protein
in cells was detected by immunoperoxidase staining. In addition,
trans- criptional activation by pMIE-expressed protein was
analyzed by cotransfection ion of IE180, the IE gene of PrV
TNL strain was cloned into the eukaryotic expression vector
pMAMneo, and constructed a recombinant expression plasmid
designated pMIE. pMIE was then transfected into the baby hamster
kidney cells ( BHK cells ), and expression of IE protein in
cells was detected by immunoperoxidase staining. In addition,
trans-criptional activation by pMIE-expressed protein was
analyzed by cotransfection with with a chloramphenicol acetyl
transferase (CAT) reporter plasmid containing the
promoter of PrV DNase gene (pDNase-CAT). The results of CAT
assay showed that
pMIE-expressed protein could increase the CAT expression about
3.4~10 fold,
which suggesting that the pMIE-expressed IE180 was functional
with ability to
transactivate the PrV early gene (DNase gene). Furthermore, to
further identify of PrV DNase gene ( pDNase-CAT ). The
results of CAT assay showed that pMIE-expressed protein could
increase the CAT expression about 3.4~10 fold, which suggesting
that the pMIE-expressed IE180 was functional with ability to
transactivate the PrV early gene ( DNase gene ). Furthermore, to
further identify the activation regions of IE180, we have
constructed various truncated mutants of IE gene including the
gradual deletionsthe activation regions of IE180, we have
constructed various truncated mutants
of IE gene including the gradual deletions of the carboxyl-
terminal coding
sequence and an in-frame deletion mutant. These mutants were
also transfected
into BHK cells respectively, and detected for their expression
of IE products by
immunoperoxidase staining. Cotransfection of these mutants
individually with nts were also transfected into BHK cells
respectively, and detected for their expression of IE products
by immunoperoxidase staining. Cotransfection of these mutants
individually with pDNase-CAT and then assayed the CAT production
by CAT-ELISA demonstrated that truncation of 271 carboxyl-
terminal amino acid residues from IE180 retained significant
function of transactivation. Further deletion of the carboxyl-
terminal region upDNase-CAT and then assayed the CAT production
by CAT-ELISA demonstrated that
truncation of 271 carboxyl-terminal amino acid residues from
IE180 retained
significant function of transactivation. Further deletion of the
carboxyl-
terminal region up to the amino acid residue 736 resulted in a
drastic loss of the activity, indicating that the amino-
terminal amino acid residues conferring transcriptional
activation. We then concluded that we have successfully
expressed functional IE180 protein by transfecting the pMIE into
BHK cells, and the N-terminal amino acid residues 1~1,189 of
IE180 are necessary for its transactivation ability.
URI: http://hdl.handle.net/11455/66277
Appears in Collections:微生物暨公共衛生學研究所

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