2A肽
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2A肽(英語:2A self-cleaving peptides)是一類長18-22個胺基酸殘基的肽片段,能誘導細胞內含有2A肽的重組蛋白自我剪切。[1][2]這種肽都有一段的序列模體,經常會在最後甘氨酸(G)和脯氨酸(P)連接處導致核糖體無法連接,從而造成「剪切」的效果。這種肽在很多科病毒中都有分布。[3][4]
目前一共有4種常用的2A肽:T2A、P2A、E2A、F2A。它們都是以來源的病毒命名的。例如第一種發現的2A肽F2A源於手足口病毒,而手足口病毒的英文名稱「foot-and-mouth disease virus」首字母是「F」,因此這種2A肽得名F2A。2A本身源自微小核糖核酸病毒科的基因命名方式。[1]
類型
[編輯]生物研究用到的2A肽目前一共有4種: P2A、E2A、F2A、T2A。其中F2A源於手足口病毒(Foot-and-mouth disease virus)、E2A源於馬甲型鼻炎病毒(Equine rhinitis A virus)、P2A源於豬捷申病毒(Porcine teschovirus)、T2A源於明脈扁刺蛾病毒(Thosea asigna virus)[1]。
下表羅列了四種2A肽的序列。雖然對2A肽的功能而言不是必要條件,在2A肽序列的N端加上一個GSG(Gly-Ser-Gly,甘氨酸、絲氨酸、甘氨酸)序列能提高2A肽誘導剪切的效率[5]。
種類 | 序列 |
---|---|
T2A | (GSG) EGRGSLL TCGDVEENPGP |
P2A | (GSG) ATNFSLLKQAGDVEENPGP |
E2A | (GSG) QCTNYALLKLAGDVESNPGP |
F2A | (GSG) VKQTLNFDLLKLAGDVESNPGP |
描述
[編輯]2A肽誘導的剪切效率較高,部分情況下,剪切效率可以達到接近100%。現有證據支持轉譯時2A肽會誘使核糖體在合成至2A肽中斷裂處的穀氨酸時,提早將前半段已合成的肽鏈放出,從而以2A肽為界形成二段多肽[6][7],但目前尚不完全清楚這一過程具體的分子機制[8][9]。
應用
[編輯]在基因工程中,2A肽可令一個開放讀框(ORF)轉譯出的肽鏈分爲數個獨立的肽鏈。如果需要令兩個蛋白分開表達(例如需要一個蛋白進入細胞核、另一個蛋白在細胞質中表達),又希望只在載體上構建一個開放讀框,可在它們的編碼區中插入一段2A肽序列。除此之外,如果兩個蛋白融合後,融合蛋白沒有功能,可以在兩個蛋白的編碼區中間插入一段編碼2A肽的序列,或將連結肽更換爲2A肽,使轉譯完成後兩個蛋白分開,獨立進行摺疊。這樣做有很大機會使兩個蛋白恢復功能[10]。
IRES同樣可以從一個開放讀框翻譯出兩個肽鏈[11],但其特性略有不同:由IRES分隔的兩段肽鏈雖在同一個轉錄本上,但因它們各自獨立轉譯,合成的肽鏈不會包含IRES序列;另外在IRES上游的肽鏈表現效率高於位在下游的肽鏈[12]。相對而言,2A肽本身的序列在剪切後仍然分別存在於上下游的二個產物肽鏈,在表現效率上,2A肽鏈上下游的肽鏈表現量相近[12]。搭配使用2A肽與IRES或是使用多個2A肽均可以在一個開放讀框中表達多個重組蛋白[13]。
不完全剪切
[編輯]不同2A肽序列造成的「剪切」效率各有不同,其中P2A最高,F2A最低。[14]以F2A連接的蛋白質有高達50%會形成融合蛋白,造成獲得新功能等意想不到的結果。[15]
參見
[編輯]參考
[編輯]- ^ 1.0 1.1 1.2 Liu, Ziqing; Chen, Olivia; Wall, J. Blake Joseph; Zheng, Michael; Zhou, Yang; Wang, Li; Ruth Vaseghi, Haley; Qian, Li; Liu, Jiandong. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Scientific Reports. 2017, 7 (1). ISSN 2045-2322. doi:10.1038/s41598-017-02460-2.
- ^ Sampath Karuna; Roy Sudipto. Live Imaging In Zebrafish: Insights Into Development And Disease. World Scientific. 2010-08-30: 51–52 [2019-01-05]. ISBN 978-981-4464-89-5. (原始內容存檔於2020-09-15).
- ^ Luke, Garry A.; de Felipe, Pablo; Lukashev, Alexander; Kallioinen, Susanna E.; Bruno, Elizabeth A.; Ryan, Martin D. Occurrence, function and evolutionary origins of ‘2A-like’ sequences in virus genomes. Journal of General Virology. 1 April 2008, 89 (4): 1036–1042. doi:10.1099/vir.0.83428-0.
- ^ Yang, X; Cheng, A; Wang, M; Jia, R; Sun, K; Pan, K; Yang, Q; Wu, Y; Zhu, D; Chen, S; Liu, M; Zhao, XX; Chen, X. Structures and Corresponding Functions of Five Types of Picornaviral 2A Proteins.. Frontiers in microbiology. 2017, 8: 1373. PMID 28785248. doi:10.3389/fmicb.2017.01373.
- ^ Kim, Jin Hee; Lee, Sang-Rok; Li, Li-Hua; Park, Hye-Jeong; Park, Jeong-Hoh; Lee, Kwang Youl; Kim, Myeong-Kyu; Shin, Boo Ahn; Choi, Seok-Yong. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PloS One. 2011, 6 (4): e18556 [2019-01-06]. ISSN 1932-6203. PMC 3084703
. PMID 21602908. doi:10.1371/journal.pone.0018556. (原始內容存檔於2020-02-27).
- ^ Donnelly, Michelle L. L.; Luke, Garry; Mehrotra, Amit; Li, Xuejun; Hughes, Lorraine E.; Gani, David; Ryan, Martin D. Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. Journal of General Virology. 2001, 82 (5): 1013–1025 [2019-01-06]. doi:10.1099/0022-1317-82-5-1013. (原始內容存檔於2019-08-04).
- ^ Luke, Garry A. Agbo, Eddy C. , 編. Translating 2A Research Into Practice. Rijeka, Croatia: Innovations in Biotechnology. 2012 [2019-01-06]. ISBN 9789535100966. OCLC 908264698. (原始內容存檔於2020-06-16).
- ^ Wang, Yuancheng; Wang, Feng; Wang, Riyuan; Zhao, Ping; Xia, Qingyou. 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori. Scientific Reports. 2015, 5 (1). ISSN 2045-2322. doi:10.1038/srep16273.
- ^ Cleavage Activity of Aphtho- and Cardiovirus 2A Oligopeptidic Sequences.. University of St Andrews. [2019-01-05]. (原始內容存檔於2016-12-30).
- ^ Szymczak-Workman, A. L.; Vignali, K. M.; Vignali, D. A. A. Design and Construction of 2A Peptide-Linked Multicistronic Vectors. Cold Spring Harbor Protocols. 2012, 2012 (2): pdb.ip067876–pdb.ip067876. ISSN 1559-6095. doi:10.1101/pdb.ip067876.
- ^ Polycistronic mRNAs and internal ribosome entry site elements (IRES) are widely used by white spot syndrome virus (WSSV) structural protein genes. Virology. 2009-05-10, 387 (2): 353–363 [2019-01-06]. ISSN 0042-6822. doi:10.1016/j.virol.2009.02.012. (原始內容存檔於2019-02-15) (英語).
- ^ 12.0 12.1 Lufkin, Thomas; Lim, Siew Lan; Ng, Patricia; Yap, Sook Peng; Kraus, Petra; Xing, Xing; V, Sivakamasundari; Chan, Hsiao Yun. Comparison of IRES and F2A-Based Locus-Specific Multicistronic Expression in Stable Mouse Lines. PLOS ONE. 2011-12-21, 6 (12): e28885 [2021-01-26]. ISSN 1932-6203. PMC 3244433
. PMID 22216134. doi:10.1371/journal.pone.0028885. (原始內容存檔於2020-07-01) (英語).
- ^ Jaenisch, Rudolf; Gao, Qing; Welstead, G. Grant; Creyghton, Menno P.; Cassady, John P.; Steine, Eveline J.; Ganz, Kibibi; Kim, Jongpil; Buganim, Yosef. Reprogramming Factor Stoichiometry Influences the Epigenetic State and Biological Properties of Induced Pluripotent Stem Cells. Cell Stem Cell. 2011-12-02, 9 (6): 588–598 [2019-01-06]. ISSN 1875-9777. PMID 22136932. doi:10.1016/j.stem.2011.11.003. (原始內容存檔於2013-05-09) (英語).
- ^ Kim, Jin Hee; Lee, Sang-Rok; Li, Li-Hua; Park, Hye-Jeong; Park, Jeong-Hoh; Lee, Kwang Youl; Kim, Myeong-Kyu; Shin, Boo Ahn; Choi, Seok-Yong. Thiel, Volker , 編. High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice. PLoS ONE. 2011-04-29, 6 (4): e18556. ISSN 1932-6203. PMC 3084703
. PMID 21602908. doi:10.1371/journal.pone.0018556.
- ^ Velychko, Sergiy; Kang, Kyuree; Kim, Sung Min; Kwak, Tae Hwan; Kim, Kee-Pyo; Park, Chanhyeok; Hong, Kwonho; Chung, ChiHye; Hyun, Jung Keun. Fusion of Reprogramming Factors Alters the Trajectory of Somatic Lineage Conversion. Cell Reports. April 2019, 27 (1): 30–39.e4. PMID 30943410. doi:10.1016/j.celrep.2019.03.023.