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標題: 豬環狀病毒第二型外殼蛋白 Cap 之結構與功能分析
Structural and Functional Analysis of the Porcine Circovirus Type 2 Capsid Protein
作者: 巫珮菁
Wu, Pei-Ching
Contributors: 黃千衿
微生物暨公共衛生學研究所
關鍵字: 豬環狀病毒第二型;外殼蛋白;間接酵素連結免疫吸附法;類病毒顆粒;密碼子最佳化;病毒顆粒組裝
Capsid protein;Recombinant subunit;ELISA;Porcine circovirus type 2 (PCV2);Capsid assembly;Virus-like particles(VLPs);Codon optimization
日期: 2012
Issue Date: 2013-11-19 12:51:07 (UTC+8)
Publisher: 微生物暨公共衛生學研究所
摘要: 豬環狀病毒第二型 (Porcine circovirus type 2, PCV2) 與離乳後豬隻的多系統性消耗症候群 (post-weaning multisystemic wasting syndrome, PMWS) 具有密切相關性,是一種豬隻新興重要疾病,會造成豬隻體重流失,並影響免疫系統,使豬隻容易受其他病原菌感染,嚴重甚至導致流產或死胎,常造成養豬業者的經濟損失。受 PMWS 影響的小豬體內組織中可分離出 PCV2 基因,而其第二開讀框 (open reading frame 2, ORF2) 基因可轉譯出病毒外殼蛋白 (viral capsid protein, 簡稱 Cap)。Cap 被認為是病毒主要的毒力蛋白,並含主要抗原決定位 (epitope),可誘發宿主免疫反應。為了測試 Cap 蛋白之抗原性,我們利用大腸桿菌 (Escherichia coli, E. coli) 表現六個 Cap 特定區域之重組次單位蛋白 (A-F) 當作抗原,以間接酵素連結免疫吸附法 (indirect enzyme-linked immunosorbent assay, indirect ELISA) 測試蛋白與畜牧場豬隻血清的反應。以12支 PCV2 陰性及26支 PCV2 陽性之特異性豬隻血清進行測定,所得之陰性血清吸光值的平均值加上3倍標準偏差 (standard deviations, S.D.) 當作陽性陰性之判定值 (cut-off value)。結果顯示所有重組蛋白做為 indirect ELISA 抗原時之特異性 (specificities) 皆為100%,但是 Cap N 端序列之 A 與 B 次單位重組蛋白對陽性血清的敏感性 (sensitivities) 不佳,而 Cap C 端的重組蛋白尤其是 C 跟 D 片段則有良好的敏感性,分別是96.2%及84.6%。利用大腸桿菌表現 Cap 重組蛋白具有簡單方便且低成本的優點,而且經由測試得知重組蛋白有良好的敏感性及特異性,顯示以 Cap 次單位重組蛋白所建立之 indirect ELISA 抗體檢測方法具有用於常規血清篩選診斷 PCV2 感染的潛力。此外,Cap 是 PCV2 唯一的結構蛋白,但是目前對於病毒顆粒組裝機制仍未明瞭,況且利用細胞培養不容易取得大量病毒進行分析研究,因此我們利用大腸桿菌大量表現 PCV2 Cap 重組蛋白,以進一步探討 Cap 蛋白序列上對於病毒顆粒組裝的重要性。透過密碼子最佳化 (codon optimization) 改善 ORF2 基因5’端序列上為大腸桿菌中少用的 arginine 密碼子,進而順利表現出全長不帶有任何 fusion tag 的 Cap 重組表現蛋白 (Cap1-233),且經電子顯微鏡觀察顯示 Cap1-233 可自行組裝成類病毒顆粒 (virus-like particles, VLPs),其形態與天然的 PCV2 病毒顆粒相似。而 N 端缺損入核訊號 (nuclear localization signal, NLS) 之 Cap51-233 與 dimerization domain 缺損的 Cap△51-103 這兩種重組蛋白都無法形成 VLPs。另外 Cap 序列上唯一的 cysteine (Cys108) 點突變之 CapC/S 重組蛋白則大部分會有不規則聚集的情況,只有少數形成 VLPs。由上述結果得知 Cap N 端1-103序列即包含 NLS 和 dimerization domain 區域是自行組裝成穩定之 VLPs 所必要的,而唯一的 Cys108 胺基酸殘基則與形成建全的 VLPs 有關。進一步利用 Cap1-233 重組蛋白進行豬隻免疫試驗,免疫豬隻在補強免疫後其抗體已顯著揚升,並在攻毒後仍健康良好且體重持續上升。相反地,對照組豬隻並無抗體反應且生長遲滯,並於攻毒後14天即死亡。綜合以上結果顯示,利用大腸桿菌表現的全長 Cap1-233 重組蛋白能自行組裝成類病毒顆粒,並能引發豬隻產生具有保護性之抗體反應,因此認為具有發展成為 PCV2 類病毒顆粒疫苗的潛力。
Porcine circovirus type 2 (PCV2) is considered to be associated with post-weaning multisystemic wasting syndrome (PMWS), which is a newly emerged economically important swine disease. The entire coding region of open reading frame 2 (ORF2), encoding the viral capsid protein (Cap), of PCV2 was cloned and sequenced from the clinical specimen obtained from PMWS-affected piglets. Six recombinant subunits, A–F, spanning the defined regions of Cap were produced by Escherichia coli (E. coli) expression system and used as antigens for testing their reactivities with swine sera in the indirect enzyme-linked immunosorbent assay (indirect ELISA). The recombinant Cap subunit-based ELISA was evaluated by examining a panel of 12 PCV2-negative and 26 PCV2-positive sera. When the positive/negative cut-off value was set at the mean value of negative sera plus 3 standard deviations, all subunits-based ELISA demonstrated 100% specificities. The N-terminal subunits, A and B, revealed poor reactivity with positive swine sera, whereas, greater immunoreactivity was observed for the C-terminal subunits of which subunits C and D demonstrated good sensitivities of 96.2% and 84.6%, respectively. The recombinant Cap subunits possessing defined antigenicity are easy to produce and the subunit-based ELISA was developed with a high specificity and sensitivity that may provide a useful method for routine serodiagnosis of PCV2 infection. Moreover, the sole structural capsid protein of PCV2, Cap, consists of major antigenic domains, but little is known about the assembly of capsid particles. The purpose of this study is to produce a large amount of Cap protein using E. coli expression system for further studying the essential sequences contributing to formation of particles. By using codon optimization of rare arginine codons near the 5′-end of the ORF2 gene for E. coli, a full-length Cap without any fusion tag recombinant protein (Cap1-233) was expressed and proceeded to form virus-like particles (VLPs) in normal Cap appearance that resembled the authentic PCV2 capsid. The N-terminal deletion mutant (Cap51-233) deleted the nuclear localization signal (NLS) domain, while the internal deletion mutant (CapΔ51-103) deleted a likely dimerization domain that failed to form VLPs. The unique Cys108 substitution mutant (CapC/S) exhibited most irregular aggregates, and only few VLPs were formed. These results suggest that the N-terminal region within the residues 1 to 103 possessing the NLS and dimerization domains are essential for self-assembly of stable Cap VLPs, and the unique Cys108 plays an important role in the integrity of VLPs. The immunogenicity of PCV2 VLPs was further evaluated by immunization of pigs followed by challenge infection. The Cap1-233-immunized pigs demonstrated specific antibody immune responses and are prevented from PCV2 challenge, thus implying its potential use for a VLP-based PCV2 vaccine.
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