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2A肽

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2A肽功能图解:编码2A肽的DNA片段插入两个蛋白的编码区中间后,可以使肽链在翻译完成后发生自剪切,分成两个独立折叠的蛋白

2A肽(英语:2A self-cleaving peptides)是一类长18-22个氨基酸残基的片段,能诱导细胞内含有2A肽的重组蛋白自我剪切。[1][2]这种肽都有一段序列模体英语sequence motif,经常会在最后甘氨酸(G)和脯氨酸(P)连接处导致核糖体无法连接,从而造成“剪切”的效果。这种肽在很多科病毒中都有分布。[3][4]

目前一共有4种常用的2A肽:T2A、P2A、E2A、F2A。它们都是以来源的病毒命名的。例如第一种发现的2A肽F2A源于手足口病毒英语foot-and-mouth disease virus,而手足口病毒的英文名称“foot-and-mouth disease virus”首字母是“F”,因此这种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]

类型

生物研究用到的2A肽目前一共有4种: P2A、E2A、F2A、T2A。其中F2A源于手足口病毒英语foot-and-mouth disease virus(Foot-and-mouth disease virus)、E2A源于马甲型鼻炎病毒(Equine rhinitis A virus)、P2A源于猪捷申病毒英语Porcine teschovirusPorcine teschovirus)、T2A源于明脉扁刺蛾病毒(Thosea asigna virus[1]

下表罗列了四种2A肽的序列。虽然对2A肽的功能而言不是必要条件,在2A肽序列的N端加上一个GSG(Gly-Ser-Gly,甘氨酸丝氨酸、甘氨酸)序列能提高2A肽诱导剪切的效率[5]

种类 序列
T2A (GSG) EGRGSLL TCGDVEENPGP
P2A (GSG) ATNFSLLKQAGDVEENPGP
E2A (GSG) QCTNYALLKLAGDVESNPGP
F2A (GSG) VKQTLNFDLLKLAGDVESNPGP

描述

2A肽诱导的剪切效率较高,部分情况下,剪切效率可以达到接近100%。现有证据支持转译时2A肽会诱使核糖体在合成至2A肽中断裂处的谷氨酸时,提早将前半段已合成的肽链放出,从而以2A肽为界形成二段多肽[6][7],但目前尚不完全清楚这一过程具体的分子机制[8][9]

应用

基因工程中,2A肽可令一个开放读框(ORF)转译出的肽链分为数个独立的肽链。如果需要令两个蛋白分开表达(例如需要一个蛋白进入细胞核、另一个蛋白在细胞质中表达),又希望只在载体上构建一个开放读框,可在它们的编码区中插入一段2A肽序列。除此之外,如果两个蛋白融合后,融合蛋白没有功能,可以在两个蛋白的编码区中间插入一段编码2A肽的序列,或将连结肽更换为2A肽,使转译完成后两个蛋白分开,独立进行折叠。这样做有很大机会使两个蛋白恢复功能[10]

IRES同样可以从一个开放读框翻译出两个肽链[11],但其特性略有不同:由IRES分隔的两段肽链虽在同一个转录本上,但因它们各自独立转译,合成的肽链不会包含IRES序列;另外在IRES上游的肽链表现效率高于位在下游的肽链[12]。相对而言,2A肽本身的序列在剪切后仍然分别存在于上下游的二个产物肽链,在表现效率上,2A肽链上下游的肽链表现量相近[12]。搭配使用2A肽与IRES或是使用多个2A肽均可以在一个开放读框中表达多个重组蛋白[13]

不完全剪切

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连接的蛋白质有高达50%会形成融合蛋白,造成获得新功能等意想不到的结果。[15]

参见

参考

  1. ^ 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. 
  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. (原始内容存档于2020-09-15). 
  3. ^ 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. 
  4. ^ 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. 
  5. ^ 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可免费查阅. PMID 21602908. doi:10.1371/journal.pone.0018556. (原始内容存档于2020-02-27). 
  6. ^ 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. (原始内容存档于2019-08-04). 
  7. ^ Luke, Garry A. Agbo, Eddy C. , 编. Translating 2A Research Into Practice. Rijeka, Croatia: Innovations in Biotechnology. 2012 [2019-01-06]. ISBN 9789535100966. OCLC 908264698. (原始内容存档于2020-06-16). 
  8. ^ 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. 
  9. ^ Cleavage Activity of Aphtho- and Cardiovirus 2A Oligopeptidic Sequences.. University of St Andrews. [2019-01-05]. (原始内容存档于2016-12-30). 
  10. ^ 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. 
  11. ^ 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. (原始内容存档于2019-02-15) (英语). 
  12. ^ 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可免费查阅. PMID 22216134. doi:10.1371/journal.pone.0028885. (原始内容存档于2020-07-01) (英语). 
  13. ^ 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. (原始内容存档于2013-05-09) (英语). 
  14. ^ 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 , 编. 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可免费查阅. PMID 21602908. doi:10.1371/journal.pone.0018556. 
  15. ^ 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.