Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89336
標題: 源自番茄斑萎病毒屬之抗原表體標定系統之開發與應用及生體內胡瓜綠斑駁嵌紋病毒載體之建立
Development and application of epitope tags system derived from the plant-infecting Tospovirus and establishment of an in-vivo Cucumber green mottle mosaic virus vector
作者: Hao-Wen Cheng
鄭浩文
關鍵字: 番茄斑萎病毒
基因沉寂抑制子
核鞘蛋白
抗原表體標定
胡瓜綠斑駁嵌紋病毒
植物病毒載體
Tospoviruses
NSs protein
Nucleocapsid protein
Epitope tag
Cucumber green mottle mosaic virus (CGMMV)
Plant viral vector
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摘要: 蛋白分子間的交互作用影響生物現象的存在與表現。隨著分子生物技術的進步,抗原表體標定可以利用DNA的重組技術連接在蛋白質上,用以偵測、或是利用針對該抗原表體的抗體進行免疫沉降實驗。所以一個高效率的抗原表體標定系統,將有助於研究生物現象背後的蛋白分子交互作用。 病毒誘導基因沈寂(Virus-induced gene silencing, VIGS)可以做為研究植物基因功能的工具,提供一個高通量的平台,來調查基因功能。病毒載體可以攜帶植物基因的部分片段,當其感染寄主植物後發生VIGS時,該相對的植物基因mRNA也會被分解,沒有蛋白產物或是表現量降低,而無法維持正常功能,導致植物產生相對應之表型(phenotypes)。目前VIGS的技術已被廣泛應用於多種寄主植物中,做基因功能調查,如植物發育、植物訊息傳導及抗病性等等。晚近,由於基因體技術進步,許多物種的全基因體序列已被完整解序。葫蘆科作物中的胡瓜、甜瓜、和西瓜等重要作物,皆已有全基因體的資料庫。因此開發專屬葫蘆科作物的VIGS病毒載體,也就成為極為重要的議題之一。 本論文的第一章前人研究分為兩個部分進行文獻回顧,第一部分為番茄斑萎病毒屬的特性以及血清學和分類上的研究介紹。第二部分則是菸草嵌紋病毒之特性和該屬病毒載體構築和外源基因表現策略的介紹。 第二章「由番茄斑萎病毒之非結構性蛋白上保留區域所得之高效率蛋白標誌應用於細菌與植物表現系統中重組蛋白表現偵測」:本實驗室長久以來投注心力於番茄斑萎病毒的偵測上,針對個別病毒蛋白製備了不同多元和單元抗體。其中NSscon monoclonal antibody (NSscon MAb) 最為特別,可偵測亞洲型番茄斑萎病毒屬病毒的NSs蛋白,其抗體辨認的抗原決定基(epitope)序列已被定位於蛋白N端,為23個胺基酸的高保留區域。由此我們也研發了與NSs-MAb相對應的抗原表體標定系統 (nss-tag),此專一性高且高力價的單株抗體和一個已知的epitope序列的相互配和與應用,可大為提升我們所製備的單株抗體的應用價值。我們以抗原決定基辨識(epitope mapping)的方式得出該epitope的有效率辨識長度可縮短至KFTMHNQIF九個胺基酸,將其應用於細菌、植物病毒、以及農桿菌表現系統中,皆可成功表現並偵測到外源蛋白。被nss-tag標定的HC-Pro可以被單株抗體偵測且基因沈寂抑制子的活性不受影響,證明nss-tag不會影響被標定蛋白質的正常功能,且nss-tag亦可被應用於免疫共沈降的實驗中來研究蛋白質間之交互作用。本章節已發表在 Journal of Biotechnology, 2013. 本論文第三章「歐美型番茄斑萎病毒和鞘蛋白上保留抗原之確認與應用」:此研究中,我們也製備了一個可以偵測到多數歐美型番茄斑萎病毒屬病毒NP蛋白的單株抗體,顯示NP蛋白上也對應有一個高保留區域,同樣可用於開發成為抗原表體標定系統 (tnp-tag)。利用細菌表達系統來表現GFP連結不同長度NP蛋白之重組蛋白來定位該epitope。可發現最小的抗體辨識區域,在NP蛋白上第211-215的位置(KGKEY),但是卻與NP抗體的反應大幅下降。由於本實驗以發展表體標定系統目的考量,因此選用六個氨基酸長度KGKEYA做為tnp-tag,目前測試結果證明tnp-tag在細菌系統中可以成功表現並偵測外源蛋白,也可以應用於免疫共沈降實驗中。其真核細胞表現系統之應用性將再進一步測試,以加強所開發的番茄斑萎病毒表體標定(tospoviral epitope tags)系統的應用性。 第四章「胡瓜綠斑駁嵌紋病毒(Cucumber green mottle mosaic virus)之全序列與生體內病毒載體之建立」: CGMMV可以感染胡瓜、甜瓜、西瓜和菸草等重要經濟作物,是一個可開發為葫蘆科作物專屬的VIGS載體的理想標的。另外,有關TMV載體建構的相關研究很多,將可應用於CGMMV載體的構築策略及參考。在本章節中,我們將CGMMV台灣分離株(CG-11)全長度的cDNA一次增幅並且選殖到二元載體上,該CGMMV的生體內感染性載體,可利用農桿菌接種在胡瓜、刺角瓜、西瓜、和煙草上,能造成與病毒感染相類似的病徵。進一步以5'RACE決定其CP subgenomic (sg) RNA的起始點為CP start codon前13個鹼基的位置。根據該位置選定sgRNA啟動子區域,將其改造成可以表現外源基因的病毒載體。利用GFP做為做為報導基因之結果顯示,CGMMV可於農桿菌接種的葉片上表現GFP蛋白,表示我們所選定的sgRNA啟動子有活性。但是此建構之CGMMV載體表現外源蛋白的效率不如預期。因此我們進一步增加不同非轉譯性鞘蛋白長度於GFP蛋白C端,發現此種方式可增進重組外源蛋白的表現。另外,為測試此載體是否具有成為VIGS載體之有效應用性,本章節也進行將外源基因片段放置在病毒的鞘蛋白與3'端非轉譯區交界處重組病毒之建構,其結果顯示,額外加入的限制酶切位對病毒沒有影響,但是放入Phytone desaturase (PDS)基因的部分片段後(300 bp),該重組病毒系統性感染寄主植物之效率不若理想,僅少數可發生系統性病徵,經解序證明其回復成野生型的病毒。由此我們發現本實驗做構築之CGMMV載體可具感染寄主之能力,且可以做為做為外源基因之病毒表現載體,但仍有許多可改進的空間,而克服外源基因表現不理想的障礙後,即可進一步開發為葫蘆科作物上的VIGS載體。 綜合以上所述,本論文主要方向為進行植物病毒單元抗體與其專一性之抗原表體標誌系統的開發,以及發展葫蘆科病毒載體的建構與應用。由於專一性高之小分子抗原表體標誌無論在基因功能及蛋白偵測上皆是許多研究人員所驥需的研究工具之一。本論文利用植物病毒蛋白序列開發了兩種小分子之蛋白標誌,目前測試顯示具有高度專一性及不會影響被標幟蛋白功能的優點,因此是一個極具學術及商品應用價值的表體標誌系統。另一方面,也開發了葫蘆科重要病毒之表達載體。雖有須更進一步修正及改進的空間,但做為未來病毒學及植物基因功能的研究,或是用於做為表現醫療疫苗或增進抗病性的應用上皆有其高度價值。此外,我們也可以進一步將nss或tnp表體標定系統與CGMMV病毒載體結合,將可有更廣泛的應用性。
The protein-protein interaction influences existence and behavior of the biological phenomenon. Epitope tagging is a technique that a known epitope is fused to recombinant proteins by genetic cloning. It is useful to characterize and monitor newly discovered proteins and existing proteins without the need for specific antibodies. One of the important applications of epitope-tagging is co-immunoprecipitaion. In this technique, a tagged-protein is immunoprecipitated with an anti-tag antibody to pull down proteins interacting directly with the tagged-protein. The power of this technique has contributed greatly to investigate protein-protein interactions involved in particular biological phenotypes. In recent years, abundant plant genome information has become available through genome sequencing analysis. Virus-induced gene silencing (VIGS) is a powerful and efficient tool to investigate gene functions in plants because of its rapidness and high-throughput. A recombinant virus for VIGS can deliver a fragment of plant gene into plant cells for inducing post-transcriptional gene silencing (PTGS) on the targeted endogenous gene to knock-down or abolish the related functions. Cucumber green mild mosaic virus (CGMMV) is a member of Tobamovirus, which infects cuburbit with high titers. The gene expression strategy of this virus makes it an ideal VIGS vector to be used for the cucurbitaceous plants. In this dissertation, Chapter 1 is the literature review describing the above relevant studies. Chapter 2 describes the development of an efficient tag derived from the common epitope of tospoviral NSs proteins for monitoring recombinant proteins expressed in both bacterial and plant systems. NSscon (23 aa), a common epitope in the gene silencing suppressor NSs proteins of the members of the Watermelon silver mottle virus (WSMoV) serogroup, was previously identified. In this investigation, green fluorescent protein (GFP)-fused with different deletions of NSscon were expressed in bacteria and reacted with NSscon monoclonal antibody (MAb). Our results indicated that the core 9 amino acids of NSscon, '109KFTMHNQIF117', denoted as 'nss', retained the reactivity of NSscon. In bacterial pET system, four different recombinant proteins labeled with nss, either at N- or C-extremes, were readily detectable without position effects, with sensitivity superior to that for the polyhistidine-tag. When the nss-tagged Zucchini yellow mosaic virus (ZYMV) helper component-protease (HC-Pro) and WSMoV nucleocapsid protein (NP) were transiently expressed by agroinfiltration in tobacco, they were readily detectable and the tag's possible efficacy for gene silencing suppression was not noticed. Co-immunoprecipitation of nss-tagged and non-tagged proteins expressed from bacteria confirmed the interaction of potyviral HC-Pro and coat protein. Thus, we conclude that this novel nss sequence is highly valuable for tagging recombinant proteins in both bacterial and plant expression systems. This chapter has been published in Journal of Biotechnology, 2013. Chapter 3 describes the identification of a conserved epitope of NP of Euro-America type tospoviruses and its application as an eitope tag for recombinant proteins. The antisera prepared against the abundant tospoviral NSs or NP in infected tissues have widely been used as the main serological tools for the detection of vital tospoviruses. The MAb 20C4C8, prepared in this study against TSWV NP, was found to be able to recognize most of the Euro-American type tospoviruses at a conserved epitope of NP. The MAb 20C4C8-recognizable short peptide '211KGKEYA216', designated tnp, trimmed from the conserved epitope of TSWV NP was used for tagging recombinant proteins expressed in the bacterial system. Using MAb 20C4C8, the tnp-tag was further applied in co-immunoprecipitation for verifying the interaction between ZYMV CP and HC-Pro. Furthermore, bacteria expressed recombinant proteins were tagged and characterized with both nss-tag and tnp-sequence, and these two tags performed amicably. Taken together, the 6 amino acid tnp-tag (KGKEYA) can be efficiently applied in bacterial expression system for monitoring recombinant proteins. Also, the tnp-tag can be coupled with the nss-tag to form a novel epitope tag system for the study of protein-protein interactions. Chapter 4 describes the elucidation of the full-length sequence and development of a Taiwan strain of CGMMV as an in vivo plant virus vector. CGMMV is a cucurbit-infecting tobamovirus, with a genome of ss(+) RNA of 6423 nucleotides. The gene expression strategy of CGMMV makes it a good candidate to be constructed as a VIGS vector for cucurbitaceous plants. Following sequencing, the full-length cDNA of CGMMV was cloned into an Agrobacterium binary vector, driven by a Cauliflower mosaic virus (CaMV) 35S promoter and terminated by a Hepatitis delta virus (HDV) ribozyme sequence for generation of the exact 3' end of CGMMV RNA. When the constructed full-length cDNA clone pk-CG was introduced into tobacco, horn melon, cucumber and watermelon plants by agroinfiltration, symptoms similar to those induced by the wild type virus were noticed and the infection was confirmed by ELISA with CGMMV antiserum. The transcription start site for the CP sub-genomic (sg) RNA was determined by 5' RACE; the result indicated that the promoter is 13 bases upstream of the start codon of CP open reading frame (ORF). The pk-CG was mutated at CP ORF (+52) by site-direct mutagenesis to create a NcoI site for in-frame insertion of the GFP ORF. The constructed CGMMV recombinant carrying the fused GFP was introduced into tobacco plants by agroinfiltration and the expression of GFP was observed in the infiltrated leaves. However, the numbers of GFP expression foci were low. The CGMMV vector with different heterologous promoters derived from other tobamoviruses expressed GFP at higher levels in the infiltrated leaves, but systemic spread was not observed. In addition, the multiple cloning sites were also created between CP ORF and 3' UTR for carrying foreign DNA fragment, but the recombinant was not stable. In conclusion, CGMMV vector is able to express GFP in the initially infected cells of infiltrated leaves and its systemic translocation remains to be further improved. Taken all together, the main purposes of this dissertation were directed to develop epitope tag system from the plant-infecting tospoviruses and to generate a cucurbit-infecting tobamovirus vector. Small epitope tags are useful to detect and analyze protein functions. The two small epitope tags from tospoviral proteins are immunodetectable with high specificity and they do not interfere with protein functions. Thus our results indicate that they are valuable for monitoring recombinant proteins. On the other hand, we have also developed a cucurbit-infecting CGMMV vector, even though the vector needs to be further improved for its efficacy as a VIGS inducer. Nevertheless, the CGMMV vector is still valuable for studying the plant-virus interaction, such as viral cell-to-cell movement in cucurbits. Finally, the two tospoviral epitope tags and the CGMMV vector can be further incorporated to increase their feasibility for many studies.
URI: http://hdl.handle.net/11455/89336
文章公開時間: 10000-01-01
Appears in Collections:植物病理學系

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