利用西瓜銀斑病毒之非結構性蛋白上的一個共同抗原表體發展可應用於細菌以及病毒蛋白表現系統上的重組蛋白標定

李建賢

doi:10.6845/NCHU.2010.01020

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利用西瓜銀斑病毒之非結構性蛋白上的一個共同抗原表體發展可應用於細菌以及病毒蛋白表現系統上的重組蛋白標定

Development of a common epitope of NSs protein of Watermelon Silver mottle tospovirus as a tag for recombinant proteins expressed in bacterial and viral systems

李建賢 , Masters　　Advisor：葉錫東　　

英文 DOI： 10.6845/NCHU.2010.01020

西瓜銀斑病毒；非結構性蛋白；抗原表體；標定； Watermelon Silver mottle tospovirus ； NSs ； tag ； epitope

Abstract │ Reference (103) │ Altmetrics

Abstract 〈TOP〉

隨著時代的變化，現代技術的發達，越來越多的蛋白質被發現且以蛋白重組技術生產出來。 藉由與親和性標定的結合，讓我們可以快速且精確的純化及分析這些重組蛋白。 但是與標定的結合可能會影響重組蛋白本身的結構及生物活性，而使用較小的胜肽標定是一種避免這種情況常用的方法。 屬於蕃茄斑萎病毒屬(Tospovirus)的西瓜銀斑病毒 (Watermelon Silver mottle virus)的S RNA上編譯了一個49.7 kDa的非結構性蛋白(Nonstructural protein )，本實驗室在先前研究中製備了NSs蛋白的單株抗體，並且證實了此單株抗體的結合位位於NSs蛋白N端的的第98至120個胺基酸的高度保留區域(NSscon)。 NSs蛋白與此單株抗體有著強烈的結合作用，此特性使這個NSscon區域有作為一個表體標定的潛力。 在本實驗中，我們將NSscon由N端或C做不同長度的移除，再將得到的不同長度片段個別的連結於綠螢光蛋白 (green fluorescent protein, GFP)的C端。 藉由西方墨點偵測(western blotting)來測試NSscon的單株抗體,對與GFP連接的不同長度片段NSscon區域的反應。 結果顯示，此單株抗體在NSscon區域上的最小辨識區域為NSs蛋白的第109至117個胺基酸109KFTMHNQIF117”，我們將此專一的辨識區域命名為”nss”。 我們更進一步的將此nss作為標定(nss-tag)，並嚐試應用於常用的pET28細菌蛋白表現系統，以及矮南瓜黃化嵌紋病毒 (Zucchini yellow mosaic virus, ZYMV)病毒載體上。 在細菌系統上同時比較偵測GFP的能力，我們的實驗結果證實，此nss-tag比現今常用的his-tag有著更強的靈敏度，而且連結於GFP的Ｎ端及Ｃ端或是同時與his-tag連結於GFP的C端，皆有著相似的靈敏度，顯示了與其他標定的複合連結及不同端點的連結並不影響nss-tag的偵測能力。 在利用矮南瓜黃化嵌紋病毒植物病毒載體系統上，分別利用與nss-tag及his-tag反應的單株抗體皆能成功的在感染的矮南瓜葉片上偵測到同時帶有此兩種標定於C端的GFP，但是同時帶有此兩種標定於C端的銀花鱸魚的生長賀爾蒙(SBGH)，可能因蛋白本身不夠穩定，無法在感染的葉片上被偵測到。 藉由結合不同特性的標定，例如nss-tag以及his-tag，可以改善單一標定功能上的不足，nss-tag可以提供較靈敏的偵測能力，而his-tag則可以提供單純簡單的純化過程。 在本實驗中，我們提供了將來在細菌載體或者是植物病毒載體的蛋白表現系統上，一個全新的高靈敏表體標定，以供重組蛋白的快速偵測。

Parallel Abstract 〈TOP〉

Nowadays, more and more proteins were produced by using modern recombinant techniques.  The affinity tags provide simple and accurate assay for the recombinant protein during purification and applicability to a number of different proteins to achieve high-throughput protein purification.  Using a very small peptide tag is a common approach to avoid the structural or functional interference on the attached proteins.  The nonstructural NSs protein (49.7 kDa) is encoded from the viral strand of Watermelon silver mottle virus (WSMoV) S RNA.  A NSs common epitope which was identified in a highly conserved region that reflects amino acid 98 to 120 of NSs proteins among tospoviruses of WSMoV serogroup.  The reaction between the monoclonal antibody and the NSs conserved region (NSscon) is strong and highly specific, indicating that NSscon could be developed as an epitope tag for detecting recombinant proteins.  In this investigation, through expressing the green fluorescent protein (GFP) with different deletions of NSscon from either the N- or C- terminal end, we identified the minimal binding motif of NSscon sequence by western blotting using the monoclonal antibody against NSscon. The essential sequence (“109KFTMHNQIF117”) of NSscon to be recognized by the MAb was denoted as “nss”.  Furthermore, we applied this nss sequence to tag recombinant proteins produced by pET28 system in E. coli and plant viral vector ZYMV in squash plants.  Our results indicated that the efficiency of detection ability with nss-tag is better than that of the his-tag in bacterial system and the detectability of the nss-tag is similar either fused at the N-terminus or the C-terminus of GFP. The combination of the nss-tag and the his-tag showed no interference with the detection ability of the nss-tag.  The GFP carried both nss-tag and his-tag was expressed in ZYMV viral vector in squash and detected by western blotting using the MAb against nss-tag or the MAb against his-tag. But the striped bass growth hormone carried both nss-tag and his-tag (SBGHnss-his) expressed in ZYMV viral vector in squash was not detected probably due to the instability of the protein.  Through the combination of different tags, nss-tag and his tag, the protein expression systems could be improved by the nss-tag for detection and the his-tag for purification.  We now provide a novel choice of a valuable epitope tag for recombinant proteins expressed in bacterial and viral systems.

Reference ( 103 ) 〈TOP〉

Arazi, T., Slutsky, S. G., Shiboleth, Y. M., Wang, Y., Rubinstein, M., Barak, S., Yang, J. and Gal-On, A. (2001). Engineering zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits. J Biotechnol, 87: 67-82.
連結：
Arnau, J., Lauritzen, C., Petersen, G. E. and Pedersen, J. (2006). Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif, 48: 1-13.
連結：
Bedouelle, H. and Duplay, P. (1988). Production in Escherichia coli and one-step purification of bifunctional hybrid proteins which bind maltose. Export of the Klenow polymerase into the periplasmic space. Eur J Biochem, 171: 541-549.
連結：
Betts, M. J. and Russell., R.B. (2003). Amino acid properties and consequences of subsitutions. In Bioinformatics for Geneticists, M.R. Barnes, I. C. Gray eds, Wiley.
連結：
Chapman, S., Kavanagh, T. and Baulcombe, D. (1992). Potato virus X as a vector for gene expression in plants. Plant J, 2: 549-557.
連結：

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