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CytoTrap酵母双杂交系统筛选突触后致密物质-93与CX3CL1相互作用位点
作者:何磊  张清秀  魏秀娥  宋加兴  荣良群 
单位:徐州医科大学, 江苏 徐州 221004
关键词:突触后致密物质-93 CX3趋化因子配体1 CytoTrap酵母双杂交 
分类号:R743
出版年·卷·期(页码):2019·38·第三期(389-396)
摘要:

目的:筛选突触后致密物质-93(PSD-93)与趋化因子CX3CL1相互作用位点。方法:对诱饵基因PSD-93(1-852aa)进行全基因合成,PCR扩增CX3CL1(1-395aa)诱饵基因全长和PSD-93-mut1(1-192aa)、PSD-93-mut2(1-420aa)、PSD-93-mut3(1-535aa)、PSD-93-mut4(1-661aa)及CX3CL1-mut(25-357aa)文库基因,并将PSD-93和CX3CL1基因重组入pSos载体,构建全长诱饵质粒pSos-PSD-93和pSos-CX3CL1;PSD-93-mut1、PSD-93-mut2、PSD-93-mut3、PSD-93-mut4、CX3CL1-mut基因被重组入pMyr载体,构建突变体质粒pMyr-PSD-93-mut1、pMyr-PSD-93-mut2、pMyr-PSD-93-mut3、pMyr-PSD-93-mut4、pMyr-CX3CL1-mut。经酶切及测序鉴定构建质粒正确后将其转化酵母菌cdc25Hα,检测其在酵母中的表达、有无自激活作用及细胞质定位,并利用CytoTrap酵母双杂交系统筛选PSD-93与CX3CL1蛋白相互作用位点。结果:成功获得重组诱饵质粒和突变体质粒,在酵母双杂交系统中,通过将构建的质粒进行两两结合,筛选出以下阳性克隆:全长质粒pSos-PSD-93+全长质粒pMyr-CX3CL1,全长质粒pSos-CX3CL1+pMyr-PSD-93-mut3,全长质粒pSos-CX3CL1+pMyr-PSD-93-mut4,从而鉴定出PSD-93和CX3CL1结合的具体氨基酸序列。结论:在本实验条件下,PSD-93和CX3CL1的结合位点分别位于PSD-93的420-535aa序列和CX3CL1的357-395aa序列。

Objective:To screen the interaction sites of the postsynaptic densitries(PSD-93) and chemokine CX3CL1. Methods:The whole gene synthesis of bait gene PSD-93(1-852aa) was conducted, and the bait gene CX3CL1(1-395aa), mut genes PSD-93-mut1(1-192aa), PSD-93-mut2(1-420aa), PSD-93-mut3(1-535aa), PSD-93-mut4(1-661aa) and CX3CL1-mut(25-357aa) were amplified by PCR. PSD-93 and CX3CL1 genes were recombined into pSos vector to construct bait plasmid pSos-PSD-93-full length and pSos-CX3CL1-full length. PSD-93-mut1, PSD-93-mut2, PSD-93-mut3, PSD-93-mut4 and CX3CL1-mut genes were recombined into pMyr vector to construct mutant plasmids pMyr-PSD-93-mut1, pMyr-PSD-93-mut2, pMyr-PSD-93-mut3, pMyr-PSD-93-mut4 and pMyr-CX3CL1-mut. After restriction enzyme digestion and sequencing, the plasmids were transformed into yeast cdc25Hα and its expression was determined in yeast. The self-activation and cytoplasmic localization were detected. The interaction between PSD-93 and CX3CL1 protein was screened by CytoTrap yeast two-hybrid system. The interaction site of PSD-93 and CX3CL1 protein was screened by CytoTrap yeast two-hybrid system. Results:The recombinant bait plasmid and mutant plasmid were obtained successfully. In the yeast two-hybrid system, the following positive clones were screened by combining the constructed plasmids, which were pSos-PSD-93-full length+pMyr-CX3CL1-full length, pSos-CX3CL1-full length+pMyr-PSD-93-mut3 and pSos-CX3CL1-full length+pMyr-PSD-93-mut4. Thus the specific amino acid sequence of PSD-93 and CX3CL1 binding was identified. Conclusion:In this experiment, the binding sites of PSD-93 and CX3CL1 are located at the 420-535aa sequence of PSD-93 and the 357-395aa sequence of CX3CL1 respectively.

参考文献:

[1] WANG J,YANG Z,LIU C,et al.Activated microglia provide a neuroprotective role by balancing glial cell-line derived neurotrophic factor and tumor necrosis factor-α secretion after subacute cerebral ischemia[J].Int J Mol Med,2013,31:172-178.
[2] CAMPANELLA M,SCIORATI C,TAROZZO G,et al.Flow cytometric analysis of inflammatory cells in ischemic rat brain[J].Stroke,2002,33(2):586-592.
[3] BUSL K M,GREER D M.Hypoxic-ischemic brain injury:pathophysiology,neuropathology and mechanisms[J].Neuro Rehabilitation,2010,26(1):5-13.
[4] ZIFF E B.Enlightening the post-synapfic density[J].Neuron,1997,19(6):1163-1174.
[5] ZHANG Q X,CHENG H,RONG R,et al.The effect of PSD-93 deficiency on the expression of early inflammatory cytokines induced by ischemic brain injury[J].Cell Biochem Biophys,2015,73(3):695-700.
[6] CHEN P,ZHAO W,GUO Y,et al.CX3CL1/CX3CR1 in Alzheimer's disease:a target for neuroprotection[J].Biomed Res Int,2016,2016:8090918.
[7] CORONA A W,HUANG Y,O'CONNOR J C,et al.Fractalkine receptor (CX3CR1) deficiency sensitizes mice to the behavioral changes induced by lipopolysaccharide[J].J Neuroinflammation,2010,7:93.
[8] ZHAO S C,MA L S,CHU Z H,et al. Regulation of microglial activation in stroke[J].Acta Pharmacol Sin,2017,38(4):445-458.
[9] LIU W M,JIANG L,BIAN C,et al.Role of CX3CL1 in diseases[J].Arch Immunol Ther Exp(Warsz),2016,64(5):371-383.
[10] SHERIDAN G K,WDOWICZ A,PICKERING M,et al.CX3CL1 is up-regulated in the rat hippocampus during memory-associated synaptic plasticity[J].Front Cell Neurosci,2014,8:233.
[11] AUDINAT E,ARNOUX I.Microglia:immune cells sculpting and controlling neuronal synapses[J].Med Sci,2014,30(2):153-159.
[12] ARNOUX I.Fractalkine signaling and microglia functions in the developing brain[J].Neural Plast,2015,2015:689404.
[13] HARRY G J,KRAFT A D.Microglia in the developing brain:a potential target with lifetime effects[J].Neurotoxicology,2012,33(2):191-206.
[14] DRINGRN R,PAWLOWSKI P G,HIRRLINGER J.Peroxide detoxification by brain cells[J].J Neurosci Res,2005,79(1-2):157-165.
[15] TANG Z,GAN Y,LIU Q,et al.CX3CR1 deficiency suppresses activation and neurotoxicity of microglia/macrophage in experimental ischemic stroke[J].J Neuroinflammation,2014,11:26.
[16] FUMAGALLI S,PEREGO C,ORTOLANO F,et al.CX3CR1 deficiency induces an early protective inflammatory environment in ischemic mice[J].Glia,2013,61(6):827-842.
[17] LAURO C,CATALANO M,TRETTEL F,et al.Fractalkine in the nervous system:neuroprotective or neurotoxic molecule?[J].Ann N Y Acad Sci,2015,1351(1):141-148.
[18] BOCKERS T M.The postsynaptic density[J].Cell Tissue Res,2006,326(2):409-422.
[19] TEGEDER I.Yeast-2-Hybrid data file showing progranulin interactions in human fetal brain and bone marrow libraries[J].Data Brief,2016,9:1060-1062.
[20] LI Q,CHEN P,ZENG Z,et al.Yeast two-hybrid screening identified WDR77 as a novel interacting partner of TSC22D2[J].Tumour Biol,2016,37(9):12503-12512.

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