Please use this identifier to cite or link to this item:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66564
Title: | 豬瘟病毒E2醣蛋白B/C區域抗原決定位之研究 Analysis of Antigenic Determinants on B/C Domains of E2 Glycoprotein of Classical Swine Fever Virus |
Authors: | Chia-Yi Chang 張家宜 |
Advisor: | 王汎熒 |
Keyword: | 豬瘟病毒,E2醣蛋白,抗原專一性,抗原決定位,單株抗體,點突變, Classical swine fever virus,E2 glycoprotein,Antigenic specificity,Conformational epitope,Monoclonal antibodies,Site-directed mutagenesis, |
Publication Year : | 2011 |
Degree: | 博士 |
Abstract: | 豬瘟為豬隻的高度傳染性疾病,常會造成感染豬隻的死亡。臺灣野外分離之豬瘟病毒株可依基因型別分為本土型 (subgroup 3.4) 及外來型 (subgroup 2.1),研究發現於1996年之後在我國田間已由外來型完全取代本土型病毒。由於懷疑不同型別病毒之間抗原性的差異可能扮演部份角色而導致病毒之間的天然置換,因E2醣蛋白位於豬瘟病毒表面為最主要誘發感染豬隻產生中和抗體之結構蛋白,因此本實驗針對不同基因型之病毒E2醣蛋白進行抗原性分析。本研究使用不同來源之抗E2單株抗體分別與不同病毒株來源之表現蛋白作用,結果顯示B/C domains在不同病毒株之E2醣蛋白具抗原專一性,可藉以區別野外株TD/96/TWN (subgroup 2.1)、94.4/IL/94/TWN (subgroup 3.4) 與疫苗株 LPC/AHRI (subgroup 1.1);而D/A domains則是較具保留性之區域。分段表現E2醣蛋白,顯示B/C domains之抗原決定位由N端90個胺基酸所組成,其構型需要693C與737C組成之雙硫鍵和motif 771LLFD774共同維持。針對表現蛋白作點突變,進一步找出713E與729D為決定野外株病毒 (94.4/IL/94/TWN) 抗原專一性之胺基酸;而705D與761K則為決定疫苗株病毒 (LPC/AHRI) 抗原專一性之胺基酸。為進一步找出B/C domains的抗原決定位,本研究使用合成之環狀胜肽模擬B/C domains的構型,其中具753RYLASLHKKALPTSV767序列的雙環胜肽,可被抗B/C domains的單株抗體以及豬隻抗豬瘟病毒的高免血清同時辨認,針對合成環狀胜肽與表達之B/C domains蛋白作點突變,進一步顯示761K,763L與764P為B/C domains的抗原決定位,而753R,754Y,755L與765T對於抗體辨認亦相當重要。因此,本研究找出一個具高度保留性的motif 753RYLASLHKKALPT765,其對於維持B/C domains的抗原結構扮演著重要角色。以上研究使我們對E2醣蛋白的抗原結構與B/C domains的抗原決定位有更深入的瞭解。 Classical swine fever (CSF), also known as hog cholera, is caused by classical swine fever virus (CSFV) and is an economically important and highly contagious disease of pigs. Analysis of the E2 sequences of CSFVs from field outbreaks in Taiwan showed that the viruses could be divided into historical subgroup (subgroup 3.4) and exotic subgroup (subgroup 2.1). In the field there has been a switch of CSFV from subgroup 3.4 to 2.1 since and after year 1996. Glycoprotein E2 of CSFV is the major antigenic protein exposed on the outer surface of the virion that induces main neutralizing antibodies during infection in pigs. To investigate whether the antigenicity differences among various CSFVs subgroups were responsible for the replacement of subgroup 3.4 by subgroup 2.1 in the field, antigenicities of E2 of various Taiwanese isolates were analyzed. This study demonstrated the antigenicity differences of E2 between vaccine and field strains of CSFV by their variable reaction patterns with monoclonal antibodies (mAbs). The mAbs against B/C domains were able to differentiate field viruses TD/96/TWN (subgroup 2.1) and 94.4/IL/94/TWN (subgroup 3.4) from the vaccine virus LPC/AHRI (subgroup 1.1). The D/A domains of various CSFVs were relatively conserved and recognized by all mAbs against the A domain. By analyszing the expressed truncated proteins, the epitope(s) on B/C domains were mapped to the N-terminal 90 residues of E2 between amino acids 690 and 779. Site-directed mutagenesis further showed that residues 693C, 737C, 771L, 772L, 773F and 774D were critical for the reactivity of E2 protein with mAbs. Thus, the B/C domains are responsible for antigen specificity among various CSFVs, and the disulfide bond and motif 771LLFD774 are essential for the structural integrity of its conformational recognition. Site-directed mutagenesis of E2 further demonstrated that residues 713E and 729D were critical for antigenic specificity of field strain (94.4/IL/94/TWN), while residues 705D and 761K were specific for vaccine strain (LPC/AHRI). These specific residues likely mediated in determining the topography of mAb binding sites of E2 to allow for differentiation between strains based on the premise that the structural integrity of the conformational epitope is maintained. To mimic the conformational epitopes, a set of synthetic cyclized peptides spanning the B/C domains of E2 were used to react with mAbs against E2 and with swine anti-CSFV polyclonal sera. All antibodies recognized a highest common element, 753RYLASLHKKALPTSV767, on the double-looped peptides. This epitope region has not been revealed previously in the literature. Both substitution-scanning of residues 753RYLASLHKKALPTSV767 on a double-looped peptide and site-directed mutagenesis of expressed E2 demonstrated that residues 761K, 763L and 764P were critical for the reactivity with mAbs. In addition, the up- and downstream residues 753R, 754Y, 755L and 765T were also crucial. Alignment showed that this stretch of amino acids was relatively conserved among various CSFVs. Thus, we identified a motif 753RYLASLHKKALPT765, which may be part of group-specific antigen and important for the structural integrity of conformational epitope recognition. These data significantly further our understanding on the antigenic structure of E2 and the antigenic dterminants on B/C domains. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66564 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 獸醫學系 |
Files in This Item:
File | Size | Format | |
---|---|---|---|
ntu-100-1.pdf Restricted Access | 1.27 MB | Adobe PDF |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.