大鼠DJ-1基因的克隆和表达
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摘要
目的与方法:帕金森(Parkinson,PD)是仅次于阿尔茨海默病的第二大常见神经变性疾病。PD疾病的发生一般以散发型为特征,被认为是基因易感性和例如暴露于毒性的外在因素的共同作用。其基因易感性也是单基因遗传性基因缺陷的结果,目前已经证实与十五种基因有关:PART1～PART11以及DAT基因、TAU基因、Pitx3基因、CTSD基因。这些基因的发现提供了一个研究PD疾病的新视角,但仍未获得对这种疾病的有效治疗措施。
DJ-1是被发现与家族性PD有关的第三个基因,蛋白质印迹分析显示DJ-1蛋白广泛存在于肝脏、骨骼肌、肾脏、大脑和睾丸。其在细胞内分布在细胞核、线粒体与胞浆,但主要在细胞浆中。在大脑组织,DJ-1蛋白在边缘系统有大量表达,这些区域包括尾状核、丘脑和黑质、海马区,这些区域与PD的关系最为密切。突变导致的DJ-1功能缺失可以诱发早发型PD,DJ-1的突变率比较低,大约1%。DJ-1蛋白在结构上属于Thij/PfPI家族,含有一个保守序列,这个家族的Thij结构与GAT结构域相关,但却有着不同的功能。DJ-1蛋白有189个氨基酸,一般以二聚体形式存在。在DJ-1晶体中,两个DJ-1单体有着广泛连接,二者接触面积覆盖了整个分子表面的35%面积,其疏水基团与功能有着密切的联系。人与猴、大鼠、小鼠、鸡的DJ-1同源性大约分别为98%、91%、91%、89%,本试验采用大鼠DJ-1作为研究。
本实验分两部分进行。第一部分为线栓法建立大鼠局灶性脑缺血再灌注模型。通过建立正确的模型,成功进行DJ-1基因的诱导表达。第二部分为大鼠DJ-1的克隆与表达。在建立模型诱导表达后,利用TRIZOL法进行RNA的提取,以DJ-1的信使RNA为模板,进行RT-PCR扩增出DJ-1编码基因。将该基因克隆入pGEM-T Easy载体,并进行DNA序列分析。在此基础上,将编码DJ-1的基因片断克隆至表达载体pQE30上,获得重组表达载体pQE30/DJ-1(简称为pQE30/DJ-1),经测序证实重组入表达载体pQE30/DJ-1的DJ-1成熟肽编码基因的序列与GenBank中所检索到的序列完全一致后,将表达载体pQE30/DJ-1转化入感受态细胞E.coli JM109,进行融合表达。表达条件进行优化后,DJ-1的表达量达到50mg/L培养基。目的蛋白含有6×His纯化标签,可以经金属鳌合亲和层析吸附于Ni-NTA树脂层析柱,梯度增加冲洗液中咪唑浓度以去除非特异吸附的杂蛋白后,将目的蛋白洗脱,超滤离心法浓缩。经SDS-PAGE、Western blotting证实蛋白质产物确实为融合蛋白DJ-1,且融合蛋白具有免疫原活性。
结果与结论:由于在国内暂时还没有DJ-1的相关研究,我们进行了DJ-1的初步探索,成功建立了原核表达系统,为进一步研究其蛋白质功能奠定了基础。并且融合蛋白的获得为以后基因工程生产有生物活性的DJ-1奠定了基础,使进一步有效治疗帕金森症成为可能。
Object and method:Parkinson's disease(PD)is the second most common neurodegenerative disorder after Alzheimer's disease.PD typically presents in a sporadic fashion and is then thought to be a result of a combination of genetic susceptibility and additional exogenous factors such as exposure to toxins.It can, however,also develop as a consequence of monogenically inherited gene defects. Fifteen PD genes have now been identified:PART1～PART11,DAT,TAU,Pitx3 and CTSD.The discovery of these PD genes has provided new insight into the pathogenesis of PD,but has not as yet led to new treatment options for this disorder.
DJ-1 is the third gene that has been linked to Parkinson disease,Northern blot analysis by Bonifati et al.showed ubiquitous expression of the DJ-1 transcript, particularly in liver,skeletal muscle,and kidney.In the brain,expression is also ubiquitous,with higher levels of the transcript in subcortical regions,such as the caudate nucleus,the thalamus,the substantia nigra,and the hippocampus,that are more affected in Parkinson disease.Mutations in the DJ-1(PARK7)gene can cause early-onset Parkinson disease(EOPD).The frequency of DJ-1 mutations in early-onset Parkinson disease is very low,at approximately 1%.Human DJ-1 belongs to ThiJ/PfpI family,whose members contain a conserved domain.The family members share a domain(ThiJ domain)that is structurally related to type I glutamine amidotransferase domain(GAT-1 domain),but the family members with known functions have distinct activities.DJ-1 is 189-amino acid protein and was also found to be a dimer.In DJ-1 crystals,two DJ-1 monomers make extensive contacts, covering 35%of the molecular surface of each molecule.The hydrophobic patches have relation with the function of DJ-1 protein.The homology of DJ-1 between human and monkey、rat、mouse、chicken is about 98%、91%、91%、89%,we use DJ-1 of rat as the material in this test.
The research is divived into two parts.In the first part,we make a focal cerebral ischemia model in rats with a suture method.After the constructing of the model,we make the success of the expressing of DJ-1.The second part is the cloning and expression of the DJ-1 encoding sequence.In this experiment,the total RNA was extracted from focal cerebral ischemia model in rats with the method of TRIZOL,and it served as the template for RT-PCR to amplify DJ-1 gene.Then DJ-1 gene was inserted into cloning vector pGEM-T easy and the gene encoding for DJ-1 was inserted into fusion expressing vector pQE-30Xa.So we get the recombinant vector pQE-30Xa/DJ-1,When the sequence was confirmed to be conformity with GenBank by the DNA sequencing,the recombinant vector pQE-30Xa/DJ-1 was transformed into E.coli JM109,and the gene was made fusion expression.After the culturing conditions were optimized,the yield of DJ-1 was 60mg/L in E.coli M15.The target proteins containing 6×His affinity tag facilitates binding to Ni-NTA resin in metal chelating affinity chromatography.After the proteins binding non-specifically to resin were removed by washing solutions with gradient increasing concentration of imidazole,the target proteins were eluted from resin with apparent higher concentration of imidazole and concentrated by centrifuge ultrafiltration.The purified fusion protein was identified by the SDS-PAGE and Western blotting,and it was confirmed to be immunogenicity.
Results and Conclusions:Because there is little research in DJ-1 gene so far in our country,we do the preliminary research and construct a prokaryotic expression system,so it will be helpful to our further research.The experiment provides a necessary basis for the preparation of bioactive mature peptide of DJ-1 by genetic engineering and makes it possible to treat PD.
DJ-1是被发现与家族性PD有关的第三个基因,蛋白质印迹分析显示DJ-1蛋白广泛存在于肝脏、骨骼肌、肾脏、大脑和睾丸。其在细胞内分布在细胞核、线粒体与胞浆,但主要在细胞浆中。在大脑组织,DJ-1蛋白在边缘系统有大量表达,这些区域包括尾状核、丘脑和黑质、海马区,这些区域与PD的关系最为密切。突变导致的DJ-1功能缺失可以诱发早发型PD,DJ-1的突变率比较低,大约1%。DJ-1蛋白在结构上属于Thij/PfPI家族,含有一个保守序列,这个家族的Thij结构与GAT结构域相关,但却有着不同的功能。DJ-1蛋白有189个氨基酸,一般以二聚体形式存在。在DJ-1晶体中,两个DJ-1单体有着广泛连接,二者接触面积覆盖了整个分子表面的35%面积,其疏水基团与功能有着密切的联系。人与猴、大鼠、小鼠、鸡的DJ-1同源性大约分别为98%、91%、91%、89%,本试验采用大鼠DJ-1作为研究。
本实验分两部分进行。第一部分为线栓法建立大鼠局灶性脑缺血再灌注模型。通过建立正确的模型,成功进行DJ-1基因的诱导表达。第二部分为大鼠DJ-1的克隆与表达。在建立模型诱导表达后,利用TRIZOL法进行RNA的提取,以DJ-1的信使RNA为模板,进行RT-PCR扩增出DJ-1编码基因。将该基因克隆入pGEM-T Easy载体,并进行DNA序列分析。在此基础上,将编码DJ-1的基因片断克隆至表达载体pQE30上,获得重组表达载体pQE30/DJ-1(简称为pQE30/DJ-1),经测序证实重组入表达载体pQE30/DJ-1的DJ-1成熟肽编码基因的序列与GenBank中所检索到的序列完全一致后,将表达载体pQE30/DJ-1转化入感受态细胞E.coli JM109,进行融合表达。表达条件进行优化后,DJ-1的表达量达到50mg/L培养基。目的蛋白含有6×His纯化标签,可以经金属鳌合亲和层析吸附于Ni-NTA树脂层析柱,梯度增加冲洗液中咪唑浓度以去除非特异吸附的杂蛋白后,将目的蛋白洗脱,超滤离心法浓缩。经SDS-PAGE、Western blotting证实蛋白质产物确实为融合蛋白DJ-1,且融合蛋白具有免疫原活性。
结果与结论:由于在国内暂时还没有DJ-1的相关研究,我们进行了DJ-1的初步探索,成功建立了原核表达系统,为进一步研究其蛋白质功能奠定了基础。并且融合蛋白的获得为以后基因工程生产有生物活性的DJ-1奠定了基础,使进一步有效治疗帕金森症成为可能。
Object and method:Parkinson's disease(PD)is the second most common neurodegenerative disorder after Alzheimer's disease.PD typically presents in a sporadic fashion and is then thought to be a result of a combination of genetic susceptibility and additional exogenous factors such as exposure to toxins.It can, however,also develop as a consequence of monogenically inherited gene defects. Fifteen PD genes have now been identified:PART1～PART11,DAT,TAU,Pitx3 and CTSD.The discovery of these PD genes has provided new insight into the pathogenesis of PD,but has not as yet led to new treatment options for this disorder.
DJ-1 is the third gene that has been linked to Parkinson disease,Northern blot analysis by Bonifati et al.showed ubiquitous expression of the DJ-1 transcript, particularly in liver,skeletal muscle,and kidney.In the brain,expression is also ubiquitous,with higher levels of the transcript in subcortical regions,such as the caudate nucleus,the thalamus,the substantia nigra,and the hippocampus,that are more affected in Parkinson disease.Mutations in the DJ-1(PARK7)gene can cause early-onset Parkinson disease(EOPD).The frequency of DJ-1 mutations in early-onset Parkinson disease is very low,at approximately 1%.Human DJ-1 belongs to ThiJ/PfpI family,whose members contain a conserved domain.The family members share a domain(ThiJ domain)that is structurally related to type I glutamine amidotransferase domain(GAT-1 domain),but the family members with known functions have distinct activities.DJ-1 is 189-amino acid protein and was also found to be a dimer.In DJ-1 crystals,two DJ-1 monomers make extensive contacts, covering 35%of the molecular surface of each molecule.The hydrophobic patches have relation with the function of DJ-1 protein.The homology of DJ-1 between human and monkey、rat、mouse、chicken is about 98%、91%、91%、89%,we use DJ-1 of rat as the material in this test.
The research is divived into two parts.In the first part,we make a focal cerebral ischemia model in rats with a suture method.After the constructing of the model,we make the success of the expressing of DJ-1.The second part is the cloning and expression of the DJ-1 encoding sequence.In this experiment,the total RNA was extracted from focal cerebral ischemia model in rats with the method of TRIZOL,and it served as the template for RT-PCR to amplify DJ-1 gene.Then DJ-1 gene was inserted into cloning vector pGEM-T easy and the gene encoding for DJ-1 was inserted into fusion expressing vector pQE-30Xa.So we get the recombinant vector pQE-30Xa/DJ-1,When the sequence was confirmed to be conformity with GenBank by the DNA sequencing,the recombinant vector pQE-30Xa/DJ-1 was transformed into E.coli JM109,and the gene was made fusion expression.After the culturing conditions were optimized,the yield of DJ-1 was 60mg/L in E.coli M15.The target proteins containing 6×His affinity tag facilitates binding to Ni-NTA resin in metal chelating affinity chromatography.After the proteins binding non-specifically to resin were removed by washing solutions with gradient increasing concentration of imidazole,the target proteins were eluted from resin with apparent higher concentration of imidazole and concentrated by centrifuge ultrafiltration.The purified fusion protein was identified by the SDS-PAGE and Western blotting,and it was confirmed to be immunogenicity.
Results and Conclusions:Because there is little research in DJ-1 gene so far in our country,we do the preliminary research and construct a prokaryotic expression system,so it will be helpful to our further research.The experiment provides a necessary basis for the preparation of bioactive mature peptide of DJ-1 by genetic engineering and makes it possible to treat PD.
引文
[1]Bonifati V,Oostra BA,Heutink P.Linking D J-1 to neurodegeneration offers novel insights for understanding the pathogene of Parkinsons disease[J].J Mol Med,2004,82:163-173
[2]Cookson MR.Pathways to Parkinsonism[J].Neuron,2003,37:7-10
[3]Valente EM,Abou-Sleiman PM,Caputo V,et al.Hereditary early-onset Parkinson's disease caused by mutations in PINK1[J].Science,2004,304:1158-1160
[4]Bandopadhyay R,Kingsbury AE,Cookson MR,et al.The expression of DJ-1(PARK7)in normal human CNS and idiopathic Parkinson' s disease[J].Brain,2004,127:420-430
[5]李晔,刘贤宇,王晓民.脑老化与神经系统退变性疾病[J].基础医学与临床,2002,3:193-199
[6]Bonifati,V Rizzu,P.van Baren,Met al.Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism.[J]Science 2003,299:256-259
[7]Abou-Sleiman,P.M.;Healy,D.G.;Quinn,N.;et al.The role of pathogenic DJ-1mutations in Parkinson's disease.[J]Ann.Neurol.2003,54:283-286
[8]Hague S,Rogaeva E,Hemandez D,Gulick C,et al.Early-onset Parkinsons disease caused by compound heterozygous DJ-1 mutation.[J]Ann.Neurol.2003,54:271-274
[9]Hedrich K,Djarmati A,Schafer N,et al.DJ-1(PARK7)mutations are less frequent than Parkin(PARK2)mutations in early-onset Parkinson disease.[J]Neurology 200462:389-394
[10]Annesi G,Savettieri G,Pugliese P,et al.DJ-1 mutations and parkinsonism-dementia-amyotrophic lateral sclerosis complex.[J]Ann.Neurol.200558:803-807
[11]MiHerDW,Ahmad R,Hague S,et al.L166Pmutant DJ-1 causative for recessive Parkinsons disease is degraded through the ubiquitin-proteasome system[J]Biol Chem,2003,2781 36588-36595
[12]刘纯青,高溪,彭岩等.蝎毒抗帕金森病小鼠多巴胺能神经元凋亡[J].基础医学与临床,2004,6:623-628
[13]周慧芳,薛冰,王晓民.免疫应答异常与中枢神经系统退变性疾病[J].基础医学与临床,2002,3:200-205
[14]Yokota T,Sugawara K,ho K,et al.Down regulation of D J-1 enhances cell death by oxidative stress,ER stress,and proteasome inhibition[J].Biochem Biophys Res Commun,2003,312:1342-1348
[15]Harding HP,Zhang Y,Zeng H,et al.An integrated stress response regulates amino acid metabolism and resistance to oxidative stress[J].Mol Cel,2003,11:619-633
[16]Rosa M.Canet-Aviles,Mark A.Wilson,David W.Miller,et al.The Parkinson's disease protein D J-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization.[J]Proc Nat Acad Sci U S A.2004 June 15;101(24):9103-9108
[17]Rosa M,Canet A,Mark A,et al.The Parkinson's disease protein D J-1 is neuro protective due to cysteine-sulfinie acid-driven mitochondrial localization[J].Proe Natl Acad Sci U S A,2004,101(24):9103-9106
[18]Junn EN,Taniguchi H,Jeong BS,et al.Interaction of DJ-1 with Daxx inhibits apoptosis signal-regulating kinase 1 activity and cell death[J].Proc Natl Acad Sci USA,2005,102(27):9691-9697
[19]臧志萍,曹晓岚,李义召。线拴法建立小鼠局灶性脑缺血再灌注模型的经验总结[J]中西医结合心脑血管病杂志,2007,June 5(6):511-514
[20]卢圣栋主编.现代分子生物实验技术(第二版)[M].中国协和医科大学出版社.1999
[21]蔡泽园.甲状腺激素受体p新同功体TRp△的初步研究.天津医科大学硕士论文.2006
[22]颜子颖,王海林译.精编分子生物学实验指南[M].科学出版社.1999
[23]黄培堂译.分子克隆实验指南第三版[M].科学出版社.2006
[24]King CH,Plikaytis BB,Shinnick TM.Isolation of plasmid DNA from mycobacteria using aresin-based alkaline lysis kit[J].Biotechniques,1995,19(3):326-330
[25]刘新宇.甲状旁腺素相关蛋白1-141和1-86的表达与生物活性测定.天津医科大学硕士论文.2005
[26]沈关心,周汝麟主编.现代免疫学试验技术(第二版)[M].湖北科学技术出版社.2002
[27]Nathan Pankratz and Tatiana Foroud.Genetics of Parkinson Disease.[J]NeuroRx.2004 April;1(2):235-242
[28]Hague S,Rogaeva E,Hernandez D,et al.Early-onset Parkinson's disease caused by compound heterozygous DJ-1 mutation.Ann Neurol 2003 54:271-274
[29]Hering R,Strauss K,M.Tao X,et al.Novel homozygous p.E64D mutation in DJ1in early onset Parkinson disease(PARK7).Hum.Mutat.2004 24:321-329.
[30]常红,黄国伟,刘莉.大鼠大脑中动脉栓塞模型的建立[J]天津医科大学学报 2007 13:327-330.
[31]谢宁,段新芬,牛英才.线栓法制备大鼠大脑中动脉脑缺血动物模型影响因素探析[J]中医药管理杂志 2006 14:51-54
[32]Shatzman AR.Expression systems.Curr.Opin.Biotechnol.1995;6:491-493
[33]Makrides SC.Strategies for achieving high-level expression of genes in Escherichia coli.Microbiol Rev.1996;60:512-538
[34]Gold,L.Expression of heterologous proteins in Escherichia coli.Methods Enzymol.1990;185:11-14
[35]张婷婷,叶波平.包涵体蛋白质的复性研究进展[J]药物生物技术 2007,14(4):306-309
[36]Smith PK,Krohn RI.Measurement of protein using bicinchoninic acid.Anal Biochem.1985;150:76
[37]Gu J,Stephenson CG,and Ladarola MJ.Recombinant proteins attached to a Ni-NTA column:Use in affinity purification of antibodies.Biotechniques.1994;17:257-262
[38]Janknecht R,de Martynoff G,Lou J,et al.Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus.Proc Natl Acad Sci USA.1991;88:8 972-8 976
[39]范云六.细菌质粒遗传及其分子生物学研究进展[J].微生物学免疫学进展,1984,(02)
[40]隋广超,胡美浩.影响大肠杆菌中外源基因表达的因素[J]生物化学与生物物理进展,1994,(02)
[41]Sun-Joo Lee,So Jung Kim,In-Kwon Kim,et al.Crystal Structures of Human DJ-1 and Escherichia coli Hsp31,Which Share an Evolutionarily Conserved Domain.J.Biol.Chem.,2003 November Vol.278,Issue 45,44552-44559
1. Vincenzo Bonifati, Ben A.Oostra and Peter Heutink. Linking DJ-1 to neurodegeneration offers novel insights for understanding the pathogenesis of Parkinson's disease. [J] Journal of Molecular Medicine (?) Springer-Verlag 2004. 120-127
2. Nathan Pankratz and Tatiana Foroud.Genetics of Parkinson Disease. [J]NeuroRx. 2004 April; 1(2): 235-242
3. Abou-Sleiman P. M, Healy D. G., Quinn N, et al.The role of pathogenic DJ-1 mutations in Parkinson's disease. [J] Ann. Neurol. 2003, 54: 283-286
4. Hering R, Strauss K, M.Tao X, et al. Novel homozygous p.E64D mutation in DJ1 in early onset Parkinson disease (PARK7). [J] Hum. Mutat. 2004, 24: 321-329
5. Bonifati V, Rizzu P, van Baren. M. J, et al.Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. [J] Science . 2003, 299: 256-259
6. Annesi G., Savettieri G, Pugliese P, et al. DJ-1 mutations and parkinsonism-dementia-amyotrophic lateral sclerosis complex. [J] Ann. Neurol. 2005. 58: 803-807,
7. Robert Hering, Karsten M.Strauss ,Xiao Tao. Novel Homozygous p.E64D Mutation in DJ-1 in Early Onset Parkinson Disease (PARK7) Published online in Wiley InterScience (www.interscience.wiley.com).
8. Rosa M. Canet-Aviles, Mark A. Wilson, David W. Miller, et al. The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization. [J] Proc Natl Acad Sci U S A. 2004 ,101(24): 9103-9108
9. Wenbo Zhou and Curt R. Freed L. DJ-1 Up-regulates Glutathione Synthesis during Oxidative Stress and Inhibits A53T (?)-Synuclein Toxicity. [J] Biol. Chem., 2005 ,Vol. 280, Issue 52,43150-43158
10. Sun-Joo Lee, So Jung Kim, In-Kwon Kim, et al.Crystal Structures of Human DJ-1 and Escherichia coli Hsp31, Which Share an Evolutionarily Conserved Domain. [J] Biol. Chem., 2003 ,Vol. 278, Issue 45, 44552-44559
11. Sourav Bandyopadhyay and Mark R Cookson. Evolutionary and functional relationships within the DJ-1 superfamily. [J] BMC Evol Biol. 2004; 4-6.
12. Eunsung Junn, Hiroyuki Taniguchi, Byeong Seon Jeong, et al. Interaction of DJ-1 with Daxx inhibits apoptosis signal-regulating kinase 1 activity and cell death. [J] Proc Natl Acad Sci U S A. 2005, July 5; 102(27): 9691-9696
13. Rosa M. Canet-Aviles, Mark A. Wilson, David W. Miller, et al. The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization. [J] Proc Nat Acad Sci U S A. 2004 June 15; 101(24): 9103-9108.
14. Marc C. Meulener, Charles L. Graves, Deepak M. Sampathu, et al .DJ-1 is present in a large molecular complex in human brain tissue and interacts with a-synuclein. [J] Journal of Neurochemistry 2005, Volume 93 1524-1529
15. Y Shinbo, T Niki, T Taira, et al. Proper SUMO-1 conjugation is essential to DJ-1 to exert its full activities. [J] Cell Death and Differentiation (2006) 13, 96-108
16. Taira T, Takahashi K, Kitagawa R, et al .Molecular cloning of human and mouse DJ-1 genes and identification of Sp1-dependent activation of the human DJ-1 promoter. Gene 263:285-292
17. Hague S, Rogaeva E, Hernandez D, et al. Early-onset Parkinson'sdisease caused by compound heterozygous DJ-1 mutation. [J] Ann Neurol 54:271-274
18. Hedrich K, Djarmati A, Schafer N, et al .(2004) DJ-1 (PARK7) mutations are less frequent than Parkin (PARK2) mutations in early-onset Parkinson disease. [J] Neurology 62:389-394
19. Patrick M, Abou-Sleiman , Daniel G. Healy ,et al. Causes of Parkinson's disease: genetics of DJ-1. 17 May 2004 / Published online: 26 June 2004 Springer-Verlag 2004
[2]Cookson MR.Pathways to Parkinsonism[J].Neuron,2003,37:7-10
[3]Valente EM,Abou-Sleiman PM,Caputo V,et al.Hereditary early-onset Parkinson's disease caused by mutations in PINK1[J].Science,2004,304:1158-1160
[4]Bandopadhyay R,Kingsbury AE,Cookson MR,et al.The expression of DJ-1(PARK7)in normal human CNS and idiopathic Parkinson' s disease[J].Brain,2004,127:420-430
[5]李晔,刘贤宇,王晓民.脑老化与神经系统退变性疾病[J].基础医学与临床,2002,3:193-199
[6]Bonifati,V Rizzu,P.van Baren,Met al.Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism.[J]Science 2003,299:256-259
[7]Abou-Sleiman,P.M.;Healy,D.G.;Quinn,N.;et al.The role of pathogenic DJ-1mutations in Parkinson's disease.[J]Ann.Neurol.2003,54:283-286
[8]Hague S,Rogaeva E,Hemandez D,Gulick C,et al.Early-onset Parkinsons disease caused by compound heterozygous DJ-1 mutation.[J]Ann.Neurol.2003,54:271-274
[9]Hedrich K,Djarmati A,Schafer N,et al.DJ-1(PARK7)mutations are less frequent than Parkin(PARK2)mutations in early-onset Parkinson disease.[J]Neurology 200462:389-394
[10]Annesi G,Savettieri G,Pugliese P,et al.DJ-1 mutations and parkinsonism-dementia-amyotrophic lateral sclerosis complex.[J]Ann.Neurol.200558:803-807
[11]MiHerDW,Ahmad R,Hague S,et al.L166Pmutant DJ-1 causative for recessive Parkinsons disease is degraded through the ubiquitin-proteasome system[J]Biol Chem,2003,2781 36588-36595
[12]刘纯青,高溪,彭岩等.蝎毒抗帕金森病小鼠多巴胺能神经元凋亡[J].基础医学与临床,2004,6:623-628
[13]周慧芳,薛冰,王晓民.免疫应答异常与中枢神经系统退变性疾病[J].基础医学与临床,2002,3:200-205
[14]Yokota T,Sugawara K,ho K,et al.Down regulation of D J-1 enhances cell death by oxidative stress,ER stress,and proteasome inhibition[J].Biochem Biophys Res Commun,2003,312:1342-1348
[15]Harding HP,Zhang Y,Zeng H,et al.An integrated stress response regulates amino acid metabolism and resistance to oxidative stress[J].Mol Cel,2003,11:619-633
[16]Rosa M.Canet-Aviles,Mark A.Wilson,David W.Miller,et al.The Parkinson's disease protein D J-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization.[J]Proc Nat Acad Sci U S A.2004 June 15;101(24):9103-9108
[17]Rosa M,Canet A,Mark A,et al.The Parkinson's disease protein D J-1 is neuro protective due to cysteine-sulfinie acid-driven mitochondrial localization[J].Proe Natl Acad Sci U S A,2004,101(24):9103-9106
[18]Junn EN,Taniguchi H,Jeong BS,et al.Interaction of DJ-1 with Daxx inhibits apoptosis signal-regulating kinase 1 activity and cell death[J].Proc Natl Acad Sci USA,2005,102(27):9691-9697
[19]臧志萍,曹晓岚,李义召。线拴法建立小鼠局灶性脑缺血再灌注模型的经验总结[J]中西医结合心脑血管病杂志,2007,June 5(6):511-514
[20]卢圣栋主编.现代分子生物实验技术(第二版)[M].中国协和医科大学出版社.1999
[21]蔡泽园.甲状腺激素受体p新同功体TRp△的初步研究.天津医科大学硕士论文.2006
[22]颜子颖,王海林译.精编分子生物学实验指南[M].科学出版社.1999
[23]黄培堂译.分子克隆实验指南第三版[M].科学出版社.2006
[24]King CH,Plikaytis BB,Shinnick TM.Isolation of plasmid DNA from mycobacteria using aresin-based alkaline lysis kit[J].Biotechniques,1995,19(3):326-330
[25]刘新宇.甲状旁腺素相关蛋白1-141和1-86的表达与生物活性测定.天津医科大学硕士论文.2005
[26]沈关心,周汝麟主编.现代免疫学试验技术(第二版)[M].湖北科学技术出版社.2002
[27]Nathan Pankratz and Tatiana Foroud.Genetics of Parkinson Disease.[J]NeuroRx.2004 April;1(2):235-242
[28]Hague S,Rogaeva E,Hernandez D,et al.Early-onset Parkinson's disease caused by compound heterozygous DJ-1 mutation.Ann Neurol 2003 54:271-274
[29]Hering R,Strauss K,M.Tao X,et al.Novel homozygous p.E64D mutation in DJ1in early onset Parkinson disease(PARK7).Hum.Mutat.2004 24:321-329.
[30]常红,黄国伟,刘莉.大鼠大脑中动脉栓塞模型的建立[J]天津医科大学学报 2007 13:327-330.
[31]谢宁,段新芬,牛英才.线栓法制备大鼠大脑中动脉脑缺血动物模型影响因素探析[J]中医药管理杂志 2006 14:51-54
[32]Shatzman AR.Expression systems.Curr.Opin.Biotechnol.1995;6:491-493
[33]Makrides SC.Strategies for achieving high-level expression of genes in Escherichia coli.Microbiol Rev.1996;60:512-538
[34]Gold,L.Expression of heterologous proteins in Escherichia coli.Methods Enzymol.1990;185:11-14
[35]张婷婷,叶波平.包涵体蛋白质的复性研究进展[J]药物生物技术 2007,14(4):306-309
[36]Smith PK,Krohn RI.Measurement of protein using bicinchoninic acid.Anal Biochem.1985;150:76
[37]Gu J,Stephenson CG,and Ladarola MJ.Recombinant proteins attached to a Ni-NTA column:Use in affinity purification of antibodies.Biotechniques.1994;17:257-262
[38]Janknecht R,de Martynoff G,Lou J,et al.Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus.Proc Natl Acad Sci USA.1991;88:8 972-8 976
[39]范云六.细菌质粒遗传及其分子生物学研究进展[J].微生物学免疫学进展,1984,(02)
[40]隋广超,胡美浩.影响大肠杆菌中外源基因表达的因素[J]生物化学与生物物理进展,1994,(02)
[41]Sun-Joo Lee,So Jung Kim,In-Kwon Kim,et al.Crystal Structures of Human DJ-1 and Escherichia coli Hsp31,Which Share an Evolutionarily Conserved Domain.J.Biol.Chem.,2003 November Vol.278,Issue 45,44552-44559
1. Vincenzo Bonifati, Ben A.Oostra and Peter Heutink. Linking DJ-1 to neurodegeneration offers novel insights for understanding the pathogenesis of Parkinson's disease. [J] Journal of Molecular Medicine (?) Springer-Verlag 2004. 120-127
2. Nathan Pankratz and Tatiana Foroud.Genetics of Parkinson Disease. [J]NeuroRx. 2004 April; 1(2): 235-242
3. Abou-Sleiman P. M, Healy D. G., Quinn N, et al.The role of pathogenic DJ-1 mutations in Parkinson's disease. [J] Ann. Neurol. 2003, 54: 283-286
4. Hering R, Strauss K, M.Tao X, et al. Novel homozygous p.E64D mutation in DJ1 in early onset Parkinson disease (PARK7). [J] Hum. Mutat. 2004, 24: 321-329
5. Bonifati V, Rizzu P, van Baren. M. J, et al.Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. [J] Science . 2003, 299: 256-259
6. Annesi G., Savettieri G, Pugliese P, et al. DJ-1 mutations and parkinsonism-dementia-amyotrophic lateral sclerosis complex. [J] Ann. Neurol. 2005. 58: 803-807,
7. Robert Hering, Karsten M.Strauss ,Xiao Tao. Novel Homozygous p.E64D Mutation in DJ-1 in Early Onset Parkinson Disease (PARK7) Published online in Wiley InterScience (www.interscience.wiley.com).
8. Rosa M. Canet-Aviles, Mark A. Wilson, David W. Miller, et al. The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization. [J] Proc Natl Acad Sci U S A. 2004 ,101(24): 9103-9108
9. Wenbo Zhou and Curt R. Freed L. DJ-1 Up-regulates Glutathione Synthesis during Oxidative Stress and Inhibits A53T (?)-Synuclein Toxicity. [J] Biol. Chem., 2005 ,Vol. 280, Issue 52,43150-43158
10. Sun-Joo Lee, So Jung Kim, In-Kwon Kim, et al.Crystal Structures of Human DJ-1 and Escherichia coli Hsp31, Which Share an Evolutionarily Conserved Domain. [J] Biol. Chem., 2003 ,Vol. 278, Issue 45, 44552-44559
11. Sourav Bandyopadhyay and Mark R Cookson. Evolutionary and functional relationships within the DJ-1 superfamily. [J] BMC Evol Biol. 2004; 4-6.
12. Eunsung Junn, Hiroyuki Taniguchi, Byeong Seon Jeong, et al. Interaction of DJ-1 with Daxx inhibits apoptosis signal-regulating kinase 1 activity and cell death. [J] Proc Natl Acad Sci U S A. 2005, July 5; 102(27): 9691-9696
13. Rosa M. Canet-Aviles, Mark A. Wilson, David W. Miller, et al. The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization. [J] Proc Nat Acad Sci U S A. 2004 June 15; 101(24): 9103-9108.
14. Marc C. Meulener, Charles L. Graves, Deepak M. Sampathu, et al .DJ-1 is present in a large molecular complex in human brain tissue and interacts with a-synuclein. [J] Journal of Neurochemistry 2005, Volume 93 1524-1529
15. Y Shinbo, T Niki, T Taira, et al. Proper SUMO-1 conjugation is essential to DJ-1 to exert its full activities. [J] Cell Death and Differentiation (2006) 13, 96-108
16. Taira T, Takahashi K, Kitagawa R, et al .Molecular cloning of human and mouse DJ-1 genes and identification of Sp1-dependent activation of the human DJ-1 promoter. Gene 263:285-292
17. Hague S, Rogaeva E, Hernandez D, et al. Early-onset Parkinson'sdisease caused by compound heterozygous DJ-1 mutation. [J] Ann Neurol 54:271-274
18. Hedrich K, Djarmati A, Schafer N, et al .(2004) DJ-1 (PARK7) mutations are less frequent than Parkin (PARK2) mutations in early-onset Parkinson disease. [J] Neurology 62:389-394
19. Patrick M, Abou-Sleiman , Daniel G. Healy ,et al. Causes of Parkinson's disease: genetics of DJ-1. 17 May 2004 / Published online: 26 June 2004 Springer-Verlag 2004
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