p38调节活化蛋白激酶绿色荧光蛋白融合载体的构建及表达

摘要/Abstract

摘要： 目的构建在哺乳动物细胞中表达的PRAK绿色荧光蛋白融合表达载体。方法将克隆在pET-14b上的PRAK亚克隆到绿色荧光蛋白载体pEGFP-C2上,随后转染Hela细胞,并在荧光显微镜下观察。结果重组质粒经酶切、PCR和测序鉴定正确无误,并在Hela细胞中得到高量表达。融合蛋白发出的绿色荧光表明EGFP-PRAK主要分布在细胞核中。结论成功构建了PRAK绿色荧光蛋白融合载体,该载体能在哺乳动物细胞中进行表达,为研究PRAK的细胞内定位和移位提供了一个重要的工具。

Abstract: Objective To construct the expression vector of p38 regulated/activated protein kinase (PRAK) fused to green fluorescent protein which is capable of expression in mammalian cells.Methods PRAK with His-tag in pET-14b expression vector was subcloned into the green fluorescent protein vector pEGFP-C2. The recombinant vector was then transfected into HeLa cells, followed by observation of the cells with fluorescent microscope.Results Identification by enzyme digestion, PCR and sequencing confirmed successful construction of the recombinant vector, which was highly expressed in HeLa cells. Green fluorescence of the fusion protein EGFP-PRAK was observed mainly in the cell nuclei.Conclusion The expression vector of PRAK fused to green fluorescent protein is successfully constructed and expressed in mammalian cells, which may facilitate the study of intracellular localization and translocation of PRAK.

中图分类号:

彭毅, 龚小卫, 邓鹏, 秦清和, 姜勇. p38调节活化蛋白激酶绿色荧光蛋白融合载体的构建及表达[J]. 南方医科大学学报, 2005, 25(03): 285-288.

PENG Yi, GONG Xiao-wei, DENG Peng, QIN Qing-he, JIANG Yong. Construction and expression of green fluorescent protein fusion vector of p38 regulated/activated protein kinase[J]. Journal of Southern Medical University, 2005, 25(03): 285-288.

参考文献

[1] Johnson GL,Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK,JNK,and p38 protein kinases[J]. Science,2002,298 (5600):1911-12.
[2] Kyriakis JM,Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation [J]. Physiol Rev,2001,81(2):807-69.
[3] 姜勇,龚小卫.MAPK信号通路对炎症反应的调控[J].生理学报,2000,52(4):267.Jiang Y,Gong XW. Regulation of inflammatory responses of MAPK signal transduction pathways [J]. Acta Physiol Sin,2000,52(4):267.
[4] Shi Y,Gaestel M. In the cellular garden of forking paths:how p38 MAPKs signal for downstream assistance[J]. Biol Chem,2002,383(10):1519-36.
[5] 龚小卫,秦清和,王静珍,姜勇.p38MAPK红色荧光蛋白融合载体的构建及表达[J].第一军医大学学报,2002,22(2):171-3.Gong XW,Qin QH,WANG JZ,et al. Construction and expression of red fluorescent protein fusion vector of p38 mitogen-activated protein kinase [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao,2002,22(2):171-3.
[6] Edlund S,Bu S,Schuster N,et al. Transforming growth factor-betal (TGF-beta)-induced apoptosis of prostate cancer cells involves Smad7-dependent activation of p38 by TGF-beta-activated kinase 1 and mitogen-activated protein kinase 3 [J]. Mol Biol Cell,2003,14(2):529-44.
[7] Bulavin,DV,Higashimoto Y,PopoffIJ,et al. Initiation ofa G2/M checkpoint after ultraviolet radiation requires p38 kinase [J].Nature,2001,411(6833):102-7.
[8] Cheng A,Chan SL,Milhavet O,et al. p38 MAP kinase mediates nitric oxide-induced apoptosis of neural progenitor cells [J]. J Biol Chem,2001,276(46):43320-7.
[9] Dash R,Schmidt AG,Pathak A,et al. Differential regulation of p38 mitogen-activated protein kinase mediates gender-dependent catecholamine-induced hypertrophy[J]. Cardiovasc Res,2003,57(3):704-14.
[10] Zhang L,Zhang L,Jiang Y,et al. Role of p38 protein kinase in heat-induced Raw cells apoptosis [J]. Chin Med J,2000,113 (4):354-7.
[11] Brancho D,Tanaka N,Jaeschke A,et al. Mechanism of p38 MAP kinase activation in vivo[J]. Genes Dev,2003,17(16):1969-78.
[12] Garcia JG,Wang P,Schaphorst KL,et al. Critical involvement of p38 MAP kinase in pertussis toxin-induced cytoskeletal reorganization and lung permeability [J]. FASEB J,2002,16(9):1064-76.
[13] Duyndam MC,Hulscher ST,van-der-Wall E,et al. Evidence for a role of p38 kinase in hypoxia-inducible factor 1-independent induction of vascular endothelial growth factor expression by sodium arsenite [J]. J Biol Chem,2003,278(9):6885-95.
[14] Zhang X,Shan P,Otterbein LE,et al. Carbon monoxide inhibition of apoptosis during ischemia-reperfusion lung injury is dependent on the p38 mitogen-activated protein kinase pathway and involves caspase 3[J]. J Biol Chem,2003,278(2):1248-58.
[15] Lim YB,Kang SS,An WG,et al. Chondrogenesis induced by actin cytoskeleton disruption is regulated via protein kinase C-dependent p38 mitogen-activated protein kinase signaling[J]. J Cell Biochem,2003,88(4):713-8.
[16] New L,Jiang Y,Han J. Regulation of PRAK subcellular location by p38 MAP kinases[J]. Mol Biol Cell,2003,14 (6):2603-16.
[17] Seternes OM,Johansen B,Hegge B,et al. Both binding and activation of p38 mitogen-activated protein kinase (MAPK) play essential roles in regulation of the nucleocytoplasmic distribution of MAPK-activated protein kinase 5 by cellular stress [J]. Mol Cell Biol,2002,22(20):6931-45.
[18] Chen G,Hitomi M,Han J,et al. The p38 pathway provides negative feedback for Ras proliferative signaling[J]. J Biol Chem,2000,275(50):38973-80.