利用酵母双杂交方法从小鼠脑cDNA文库中筛选朊蛋白的互作蛋白
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摘要
传染性海绵状脑病(TSE)是由异常朊蛋白(或称朊病毒)PrPsc引起的人和动物的一组中枢神经系统变性疾病。PrPsc是由富含α-螺旋的正常朊蛋白PrPc转变而来,PrPsc富含β-折叠,并具有蛋白酶抗性等特征。PrPc是一个靠糖基磷脂酰肌醇锚锚定的高度保守的蛋白质,主要在神经元表达。PrPc向PrPsc转变的分子机制及TSE发病机理至今仍不清楚,研究表明体内存在某种重要蛋白影响PrPc向PrPsc转变,所以,PrPc的互作蛋白筛选研究,将有助于PrPc向PrPsc转变、TSE发病机理及PrPc转变的生理学功能的理解。
目前,用于筛选蛋白质的互作蛋白技术很多,而酵母双杂交系统从建立至今己成为筛选蛋白质的互作蛋白强有力的方法之一。CytoTrap酵母双杂交系统采用了一个独特的酵母菌株cdc25H,该基因表达的cdc25突变体抑制其在37℃生长,但允许其在25℃温度生长。这个突变基因失去的功能可以由hSos基因来补充:通过蛋白质之间的相互作用,hSos蛋白可以定位到细胞膜上并激活Ras途径,使得cdc25H菌株可以在37℃生长。由于所有蛋白间的相互作用都发生在细胞质中,所以这个系统可以称作是酵母细胞质双杂交系统。它克服了传统酵母双杂交系统的局限性,扩展了酵母双杂交的应用范围。
本实验把诱饵质粒pSos-prp-23-231转入酵母菌cdc25Hα,检测其表达产物在酵母细胞中毒性作用和自激活作用,并利用该诱饵质粒从小鼠脑cDNA文库中筛选PrP的互作蛋白,实验结果表明:构建诱饵质粒pSos-prp-23-231可以用来钓取PrP的互作蛋白,并从小鼠脑cDNA文库中筛选出PrP的10个互作的克隆,进一步验证表明,9个为假阳性克隆,1个为阳性克隆。阳性克隆提取质粒并测序分析表明,PrP互作蛋白为Ras蛋白特异鸟嘌呤核苷酸释放因子1(Ras-grf1),利用免疫共沉淀方法进一步证明筛选出的Ras-grf1与PrP的互作关系。PrP互作蛋白Ras-grf1的发现将有助于PrPc向PrPsc转变机制及PrP生理功能的研究。
Transmissible spongiform encephalopathy (TSE) is a human and animal’s central nervous system degenerative disease. The typical feature is the acumulation of PrPsc which is abound with theβ-fold and possesses partial protease resistance according to the histopathology. PrPsc is from the PrPc that is abound withα-Helix, which mechanism is still unknown. PrPc is a high conservative protein that anchors to the cell membrane surface by glycosyl-phosphatidyl inositol anchor (GPI anchor) most expressed in neuron.Some researchers presume that there may be some other proteins affect in the pathogenesy of TSE, so searching for the proteins interacted with PrPc is significant for the research of the transform mechanism, and also the physiology function of PrPc.
There are several technologies can be used to study protein-protein interaction, Yeast two-hybrid system has became one of the most drastic methods in the study of protein-protein interaction.The CytoTrap system uses the yeast S. cerevisiae temperature-sensitive mutant strain cdc25H,The cdc25 mutation present in the cdc25H strain prevents growth at 37℃, but allows normal growth at the permissive temperature (25℃). The CytoTrap system is based on the ability of the human Sos protein (hSos), to complement the cdc25 defect and to activate the yeast Ras-signaling pathway. Expression of hSos and its subsequent localization to the plasma membrane allows the cdc25H yeast strain to grow at 37℃. The localization of hSos to the plasma membrane occurs through the interaction of two-hybrid proteins. All the interactions of protein-protein happen in cytoplasm, so it can overcome the limitations of the traditionary yeast two-hybrid system.
The prion gene subcloned into the pSos plasmid. The expression of the fusion protein PrP23-231 was not toxic to cdc25H and does not have self-activation were detected through transformation of the bait plasmid. The Cyto Trap yeast two-hybrid system could be used to fish the interacting proteins with PrP23-231 domain of mouse prion;At last, 1 positive clone was picked up among 10 putative positive clones after verification. By the extracting the plasmid from the positive clones, endonucleases digestion analysis and sequencing, it showed that Ras protein-specific guanine nucleotide-releasing factor 1(Ras-grf1).In order to validate interaction between protein Ras-grf1 and PrP23-231, we amplified co-immunoprecipitation to further validate interaction. The discoveries of the interaction protein may have important significances for the research of the transform mechanism of PrPc to PrPsc and elucidating the physiology function function of PrP.
目前,用于筛选蛋白质的互作蛋白技术很多,而酵母双杂交系统从建立至今己成为筛选蛋白质的互作蛋白强有力的方法之一。CytoTrap酵母双杂交系统采用了一个独特的酵母菌株cdc25H,该基因表达的cdc25突变体抑制其在37℃生长,但允许其在25℃温度生长。这个突变基因失去的功能可以由hSos基因来补充:通过蛋白质之间的相互作用,hSos蛋白可以定位到细胞膜上并激活Ras途径,使得cdc25H菌株可以在37℃生长。由于所有蛋白间的相互作用都发生在细胞质中,所以这个系统可以称作是酵母细胞质双杂交系统。它克服了传统酵母双杂交系统的局限性,扩展了酵母双杂交的应用范围。
本实验把诱饵质粒pSos-prp-23-231转入酵母菌cdc25Hα,检测其表达产物在酵母细胞中毒性作用和自激活作用,并利用该诱饵质粒从小鼠脑cDNA文库中筛选PrP的互作蛋白,实验结果表明:构建诱饵质粒pSos-prp-23-231可以用来钓取PrP的互作蛋白,并从小鼠脑cDNA文库中筛选出PrP的10个互作的克隆,进一步验证表明,9个为假阳性克隆,1个为阳性克隆。阳性克隆提取质粒并测序分析表明,PrP互作蛋白为Ras蛋白特异鸟嘌呤核苷酸释放因子1(Ras-grf1),利用免疫共沉淀方法进一步证明筛选出的Ras-grf1与PrP的互作关系。PrP互作蛋白Ras-grf1的发现将有助于PrPc向PrPsc转变机制及PrP生理功能的研究。
Transmissible spongiform encephalopathy (TSE) is a human and animal’s central nervous system degenerative disease. The typical feature is the acumulation of PrPsc which is abound with theβ-fold and possesses partial protease resistance according to the histopathology. PrPsc is from the PrPc that is abound withα-Helix, which mechanism is still unknown. PrPc is a high conservative protein that anchors to the cell membrane surface by glycosyl-phosphatidyl inositol anchor (GPI anchor) most expressed in neuron.Some researchers presume that there may be some other proteins affect in the pathogenesy of TSE, so searching for the proteins interacted with PrPc is significant for the research of the transform mechanism, and also the physiology function of PrPc.
There are several technologies can be used to study protein-protein interaction, Yeast two-hybrid system has became one of the most drastic methods in the study of protein-protein interaction.The CytoTrap system uses the yeast S. cerevisiae temperature-sensitive mutant strain cdc25H,The cdc25 mutation present in the cdc25H strain prevents growth at 37℃, but allows normal growth at the permissive temperature (25℃). The CytoTrap system is based on the ability of the human Sos protein (hSos), to complement the cdc25 defect and to activate the yeast Ras-signaling pathway. Expression of hSos and its subsequent localization to the plasma membrane allows the cdc25H yeast strain to grow at 37℃. The localization of hSos to the plasma membrane occurs through the interaction of two-hybrid proteins. All the interactions of protein-protein happen in cytoplasm, so it can overcome the limitations of the traditionary yeast two-hybrid system.
The prion gene subcloned into the pSos plasmid. The expression of the fusion protein PrP23-231 was not toxic to cdc25H and does not have self-activation were detected through transformation of the bait plasmid. The Cyto Trap yeast two-hybrid system could be used to fish the interacting proteins with PrP23-231 domain of mouse prion;At last, 1 positive clone was picked up among 10 putative positive clones after verification. By the extracting the plasmid from the positive clones, endonucleases digestion analysis and sequencing, it showed that Ras protein-specific guanine nucleotide-releasing factor 1(Ras-grf1).In order to validate interaction between protein Ras-grf1 and PrP23-231, we amplified co-immunoprecipitation to further validate interaction. The discoveries of the interaction protein may have important significances for the research of the transform mechanism of PrPc to PrPsc and elucidating the physiology function function of PrP.
引文
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[2]周剑涛,潘秋香.疯牛病病原体的研究进展[J].生物学通报,2001(8):16-17.
[3]南木甲,商营利,刘思当.朊病毒及疯牛病[J].山东畜牧兽医,2001(11):302-308.
[4] Prusiner S B.Novel proteinaceous infectious particles cause scrapie[J].Science,1982 (216):136-144.
[5] Prusiner S B. Molecular biology and pathogenesis of Prion diseases[J].TIBS,1996(21):482-487.
[6] Acutis PL,Bossers A,Priem J,Riina MV,et al.Identification of prion protein gene polymorphisms in goats from Italian scrapie outbreaks[J].J Gen Virol,2006 (87):1029-1033.
[7] Chavez LS,Serda R,Choe S,et al.Molecular basis for pseudo vitamin D-deficiency rickets in the Hannover pig[J].J Nutr Biochem,2003(14):378-385.
[8] Agyemang K.Trypano tolerant live stock in the context of trypanosomiasis intervention strategies [J].PAAT Technical and Scientific Series,2005(7):11-15.
[9] Gossert AD,Bonjour S,Lysek DA,et al.Prion protein NMR structures of elk and of mouse/elk hybrids[J].Proc.Natl.Acad.Sci.USA2005(102):646-650.
[10] Hornemann S,Schorn C,Wüthrich K,et al.NMR structure of the bovine prion protein isolated from healthy calf brains[J].EMBO Rep,2004 (5):1159-1164.
[11] Jeffrey M,Goodsir CM,Race RE,et al.Scrapie-specific neuronal lesions are independent of neuronal PrP expression[J].Ann Neurol 2004(55):781-792.
[12] Masel J,Genoud N,Aguzzi A.Efficient inhibition of prion replication by PrP-Fc(2) suggests that the prion is a PrP(Sc) oligomer[J].J Mol Biol 2005(345):1243-1251.
[13] Barrett JC,Fry B,Maller J,et al.Haploview:analysis and visualization of LD and haplotype maps [J].Bioinformatics,2005(21):263-635.
[14]方元.朊病毒研究进展[J].病毒学报,2000 (23):378-382.
[15] Arima K,Nishida N,Sakaguchi S,et al.Biological and biochemical characteristics of prion strains conserved in persistently infected cell cultures[J].Virol,2005 (79):7104-7112.
[16] Bergstr?m AL,Cordes H,Zsurger N,et al.Amidation and structure relaxation abolish the neurotoxicity of the prion peptide PrP106-126 in vivo and in vitro[J].Biol.Chem,2005 (280):23114-23121.
[17] Coulpier M,Messiaen S,Hamel R,et al.Bax deletion does not protect neurons from BSE-induced death[J].Neurobiol.Dis,2006 (23):603-611.
[18] Massimino ML,Griffoni C,Spisni E,et al.Involvement of caveolae and caveolae-like domains in signalling[J].cell survival and angiogenesis,2002(2):93-98.
[19] Williams TM , Lisanti MP . Caveolin-1 in oncogenic transformation , cancer , and metastasis[J].American Journal of Physiology-Cell Physiology,2005(3):494-506.
[20] Rivera-Milla E,Oidtmann B,Panagiotidis CH,et al.Disparate evolution of prion protein domains and the distinct origin of Dop-pel and prion-related loci revealed by fish-to-mammal comparisons[J].FASEB J,2006(20):317-319.
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