酵母双杂交筛选禽流感病毒PA相互作用蛋白的研究
摘要
禽流感病毒是正黏病毒科的主要成员,是单股负链分节段的RNA病毒,依赖自身的RNA聚合酶复合体催化其基因组在宿主细胞核中进行转录和复制。目前多种研究报道表明禽流感病毒能够跨种传播,给人类健康造成了巨大的威胁,因此加强禽流感病毒致病机制的研究显得格外重要。
聚合酶酸性蛋白(PA)是RNA聚合酶复合体3个亚基中的重要角色,它不但参与病毒复制、病毒RNA转录、病毒粒子组装等多种病毒活动过程,还具有核酸内切酶活性和蛋白酶活性。本文以高致病性禽源H5N1亚型禽流感病毒PA原核表达及相互作用蛋白筛选为切入点,对PA蛋白的功能进行了初步研究。
首先,构建了17个PA突变体的表达载体,SDS-PAGE电泳及Western blot分析突变体的表达丰度,证明PA基因的61-120bp和325-426bp可能是影响PA蛋白表达的两个重要区域,同时N端缺失长度在143-408个氨基酸残基之间的9个突变体在大肠杆菌中获得高表达。为制备各片段的特异性抗血清、验证蛋白的相互作用区域、表达得到全长PA蛋白奠定基础。
其次,采用MATCHMAKER GAL4 Two-hybrid System 3(clontech公司),以H5N1型病毒的PA为诱饵,筛选人肝cDNA文库相互作用蛋白,得到8个蛋白与PA相互作用,分别为GPATCH8、GPR89A、ADH1A、ADH1C、TTR、PMSA、LBP、ETFA,并在酵母内验证这6个蛋白与H5N1型病毒的PA有相互作用。
第三,根据GenBank相关序列资料设计特异引物,通过PCR的方法,扩增得到GPR89A、ADH1C、TTR、PMSA、LBP、ETFA6个全长基因。构建了6个蛋白的原核及真核表达载体,Western blot检测,除GPR89A外其余蛋白在肺腺癌细胞A549中有表达。为后续免疫共沉淀试验进一步验证蛋白相互作用奠定基础。
本研究发现了影响PA原核表达的区域,并筛选出8种PA的相互作用蛋白,进一步揭示了禽流感病毒致病过程中PA蛋白的功能,促进了对流感病毒致病机理的认识,为禽流感病毒疫苗和新型抗病毒药物的研究提供依据。
The molecular determinants and related mechanisms that make certain influenza viruses highly pathogenic species,including humans,remain poorly understood.Numerous studies have shown that influenza virus virulence in mammalian species is a polygenic trait.
The influenza virus RNA polymerase is a complex composed of three subunits,PA,PB1, and PB2.The polymerase,together with the nucleoprotein(NP) and the viral RNA template, (RNP),which carry out viral transcription and replication.PA not only carry out viral transcription and replication,but also induces a generalized proteolytic process.In this paper, the expression of PA protein and the protein interacting with PA were studied.
First,17 deletion mutants of PA protein were constructed and expressed in Ecoli.Rosseta GamiB(DE),we conclusion that the two segments of 61th-120th bp and the 325th-426th bp of PA gene may be the very fragments that affect the expression level of PA protein.At the same time,nine mutants with the deletion between the 1th bp and the 426th bp of the PA gene got higer expression.
Second,we use H5N1 PA as bait protein to screen the related protein by yeast Two-hybrid System 3.Eight positive clones were identified,they are GPATCH8、GPR89A、ADH1A、ADH1C、TTR、PMSA、LBP、ETFA.The interaction of these 8 proteins with PA were tested by yeast mating.
Third,the full-length of gene GPR89A、ADH1C、TTR、PMSA、LBP and ETFA are cloned and constructed into pCMV-3HA vector and pTIG-Trx-Etag vector.Protein ADH1C、TTR、PMSA、LBP and ETFA can be expressed by A549.
In conclusion,based on these results,the function of PA protein and the molecular pathogenic mechanism of AIV could be greatly and deeply enderstood,and the strategy for prevention against and treatment for AIV would be formed.
聚合酶酸性蛋白(PA)是RNA聚合酶复合体3个亚基中的重要角色,它不但参与病毒复制、病毒RNA转录、病毒粒子组装等多种病毒活动过程,还具有核酸内切酶活性和蛋白酶活性。本文以高致病性禽源H5N1亚型禽流感病毒PA原核表达及相互作用蛋白筛选为切入点,对PA蛋白的功能进行了初步研究。
首先,构建了17个PA突变体的表达载体,SDS-PAGE电泳及Western blot分析突变体的表达丰度,证明PA基因的61-120bp和325-426bp可能是影响PA蛋白表达的两个重要区域,同时N端缺失长度在143-408个氨基酸残基之间的9个突变体在大肠杆菌中获得高表达。为制备各片段的特异性抗血清、验证蛋白的相互作用区域、表达得到全长PA蛋白奠定基础。
其次,采用MATCHMAKER GAL4 Two-hybrid System 3(clontech公司),以H5N1型病毒的PA为诱饵,筛选人肝cDNA文库相互作用蛋白,得到8个蛋白与PA相互作用,分别为GPATCH8、GPR89A、ADH1A、ADH1C、TTR、PMSA、LBP、ETFA,并在酵母内验证这6个蛋白与H5N1型病毒的PA有相互作用。
第三,根据GenBank相关序列资料设计特异引物,通过PCR的方法,扩增得到GPR89A、ADH1C、TTR、PMSA、LBP、ETFA6个全长基因。构建了6个蛋白的原核及真核表达载体,Western blot检测,除GPR89A外其余蛋白在肺腺癌细胞A549中有表达。为后续免疫共沉淀试验进一步验证蛋白相互作用奠定基础。
本研究发现了影响PA原核表达的区域,并筛选出8种PA的相互作用蛋白,进一步揭示了禽流感病毒致病过程中PA蛋白的功能,促进了对流感病毒致病机理的认识,为禽流感病毒疫苗和新型抗病毒药物的研究提供依据。
The molecular determinants and related mechanisms that make certain influenza viruses highly pathogenic species,including humans,remain poorly understood.Numerous studies have shown that influenza virus virulence in mammalian species is a polygenic trait.
The influenza virus RNA polymerase is a complex composed of three subunits,PA,PB1, and PB2.The polymerase,together with the nucleoprotein(NP) and the viral RNA template, (RNP),which carry out viral transcription and replication.PA not only carry out viral transcription and replication,but also induces a generalized proteolytic process.In this paper, the expression of PA protein and the protein interacting with PA were studied.
First,17 deletion mutants of PA protein were constructed and expressed in Ecoli.Rosseta GamiB(DE),we conclusion that the two segments of 61th-120th bp and the 325th-426th bp of PA gene may be the very fragments that affect the expression level of PA protein.At the same time,nine mutants with the deletion between the 1th bp and the 426th bp of the PA gene got higer expression.
Second,we use H5N1 PA as bait protein to screen the related protein by yeast Two-hybrid System 3.Eight positive clones were identified,they are GPATCH8、GPR89A、ADH1A、ADH1C、TTR、PMSA、LBP、ETFA.The interaction of these 8 proteins with PA were tested by yeast mating.
Third,the full-length of gene GPR89A、ADH1C、TTR、PMSA、LBP and ETFA are cloned and constructed into pCMV-3HA vector and pTIG-Trx-Etag vector.Protein ADH1C、TTR、PMSA、LBP and ETFA can be expressed by A549.
In conclusion,based on these results,the function of PA protein and the molecular pathogenic mechanism of AIV could be greatly and deeply enderstood,and the strategy for prevention against and treatment for AIV would be formed.
引文
[1]付朝阳.禽流感相关的科技术语[J].科技术语研究,2004,6(1):45-48
[2]甘孟侯.禽流感[M].北京:北京农业大学出版社,1995
[3]乔传玲,于康震.禽流感病毒结构蛋白及其免疫原性[J].预防兽医学进展,2000,2(4):4-7
[4]陈化兰,丁康震,卢景良.禽流感病毒及其分子生物学研究进展[J].国外兽医学畜禽传染病,1997,17(1):3-7
[5]Li K S,Guan Y,Wang J,Smith G J,etal.Genesis of a highly pathogenic and potentially pandemic H5N1influenza virus in eastern Asia[J].Nature,2004,430(8):209-213
[6]Kuiken,T,Rimmelzwaan G,Baars M,etal.Avian H5N1 influenza in cats[J].Science,2004,306(10):241
[7]Puthavathana,P,Auewarakul P,Charoenying P C,etal.Molecular characterization of the complete genome of human i(?)fluenza H5N1 virus isolates from Thailand[J].J Gen.Virol,2005,86(2):(?)23-433
[8]Mongkol Uiprasertkul,Pilaipan Puthavathana,Kantima Sangsiriwut,etal.Influenza A H5N1 replication sites in humans[J].Emerg.Infect.Dis,2005,11(7):1036-1041
[9]To KF,Chan P K,Chan K F,etal.Pathology of fatal human infection associated with avian influenza A H5N1 virus[J].J Med.Virol,2001,63(3):242-246
[10]Govorkova,E A,Rehg J E,Krauss S,etal.Lethality to Ferrets of H5N1 influenza viruses isolated from humans and poultry in 2004[J].J Virol,2005,79(4):2191-2198
[11]金奇.医学分子病毒学(第一版)[M].北京:中国农业出版社,2002
[12]Matrosovich MN,Matrosovich TY,Gray T,etal.Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium[J].J Virol,2004,78(22):12665-7
[13]Gocnik M,Fislova T,Sladkova T,etal.Antibodies specific to the HA2 glycopolypeptide of influenza A virus haemagglutinin with fusion-inhibition activity contribute to the protection of mice against lethal infection[J].J Gen Virol.2007,88(3):951-955
[14]Shahidi M,Kheiri MT,Amini B O S,etal.Molecular and phylogenetic analysis of human influenza virus among Iranian patients in Shiraz[J].Iran.J Med Virol.2007,79(6):803-810
[15]Lin D,Lan J,Zhang Z.Structure and function of the NS1 protein of influenza A virus[J].Acta Biochim Biophys Sin.2007,39(3):155-162
[16]Bornholdt ZA,Prasad BV.X-ray structure of the virus NS1 effector domain[J].Nat Struct Mol Biol.2006 13(6):559-560
[17]Hale BG,Randall RE.PI3K signaling during influenza A virus infections[J].Biochem Soc Trans.2007,35(2):186-187
[18]Hatada E,Saito S,Fukuda R.Mutant influenza viruses with a defective NS1 protein cannot block the activation of PKR in infected cell[J].J Virol,1999,73(3):2425-2433
[19]Muster T,Rajtarova J,Sachet M,etal.Bergmann M.interferon resisitance promotes oncolysis by influenza virus NS1-deletion mutants[J].Int J Cancer.2004 110(1):15-21
[20]Wang XY,Li M,Zheng HY,etal.Influenza A virus NS1 protein Prevents Activation of NF-B and induction of alpha/beta interferon[J].J Virol,2000,74(24):11566-11573
[21]Skehel J J,Wiley D C.Receptor binding and membrane fusion in virus entry:the influenza hemagglutinin[J].Annu Rev Biochem,2000,69(7):531-569
[22]Nayak D P,Hui E K,Barman S.Assembly and budding of influenza virus[J].Virus Res,2004,106(2):147-165
[23]陶攀,潘兹书,吴建国.A型流感病毒RNA聚合酶及其对基因组复制和转录的调控作用[J].中国病毒学,2006,21(3):304-308
[24]Maite Huarte,Ana Falco(?)n,Yuri Nakaya,etal.Threonine 157 of Influenza Virus PA Polymerase Subunit Modulates RNA Replication in Infectious Viruses[J].J Virol,2003,77(10):6007-6013
[25]JJ Sanz-Ezquerro,S.de la Luna,J.Ort(?)n,etal.Individual expression of influenza virus PA protein induces degradation of coexpressed proteins[J].J.Virol,1995,69(2-3):2420-2426
[26]Hara K.,Shiota M,Kido H,etal.Influenza virus RNA polymerase PA subunit is a novel serine protease with Ser624 at the active site[J].Genes Cells,2001,6(2):87-97
[27]JJ Sanz-Ezquerro,J.F(?)rnandez-Santar(?)n,T Sierra,etal.The PA influenza virus polymerase subunit is a phosphorylated protein[J].J.Gen.Virol,1998,79:471-478
[28]Perales B,Sanz-Ezquerro,P Gastaminza,etal.The replication activity of influenza virus polymerase is linked to the capacity of the PA subunit to induce proteolysis[J].J.Virol,2000,74(3):1307-1312
[29]Sanz-Ezquerro,J.J.,T.Z(u|¨)rcher etal.The amino-terminal one-third of the influenza virus PA protein is responsible for the induction of proteolysis[J].J.Virol.1996,70(3):1905-1911
[30]Li ML,Rao P,Robert MK.The acive sites of the influnenza cap-dependent endonuclease are on different polymerase subunits[J].the EMBO 2001,20(8):2078-2086
[31]He X J,Zhou J,Mark Bartlam,etal.Crystal structure of the polymerase PA_C-PB1_N complex from an avian influenza H5N1 virus[J].nature Letters,2008 09
[32]Hooker L,Strong R,Admas R.A sensitive,signle-tube assay to measure the enzymatic activities of influenza RNA polymerase and other poly(A) polymerase:application to kinetic and inhibitor analysis [J].Nucleic Acides Res.2001,29(13):2691-2698
[33]Fechter P,Mingay L,Sharps J,etal.Tow aromatic residues in the PB2 subunit of influenza A RNA polymerase are curcial for cap binding[J].J Biol Chem,2003,278(22):20381-20388
[34]赵宏宇,赵育莹,曲章义,等.流感病毒RNA聚合酶亚基PA6片断的克隆及表达[J].国外医学免疫学分册,2004,27(6):362-364
[35]乔伟振,孟继鸿,董晨,等.HCV不同编码区重组基因的蛋白表达丰度分析[J].南京医科大学学报自然科学版,2008,28(6):722-725
[36]Mikulasova A,Vareckova E,and Fodor E.Transcription and replication of the influenza a virus genome[J].Acta Virol,2000,44(5):273-282
[37]Mei-Ling Li,Ping Rao and Robert M.Krug.The active sites of the infuenza cap-dependent endonuclease are on different polymerase subunits[J].The EMBO Journal 2001,20(8):2078-2086
[38]Imen R E,Amine S,Noureddine K,etal.A strategy for high-level expression of soluble and functional human interferon a as a GST-fusion protein in E.coli[J].Protein Engineering,Design & Selection.2007,20(4):201-209
[39]Imen R E,Amine Sadok,Noureddine Khalaf,etal.A strategy for high-level expression of soluble and functional human interferon a as a GST-fusion protein in E.coli[J].Protein Engineering,Design &Selection,2007,12(4):1-9
[40]谢荣辉,朱函坪,翁景清,等.流行性乙型脑炎病毒囊膜蛋白截片段的克隆及表达[J].中国计划免疫,2007,13(6):533-535
[41]Sandro De Falco,Maria Grazia Ruvoletto,Antonio Verdoliva.Cloning and Expression of a Novel Hepatitis B Virus-binding Protein from HepG2 Cells[J].the J of Bio Chemi,2007,276(39):36613-36623
[42]Siigur J,Samel M,Tonismagi K,etal.Biochemical characterization of Lebetase,a direct-acting fibrinolytic enzyme from Vipera Lebetina snake venom[J].Thromb Res,1998,90(1):39-49
[43]李佰良.功能蛋白质组学[J].生命的化学,1998,18(6):1-3
[44]Jeremy Lubana,Stephen P Goff.The yeast two-hybrid system for studying protein-protein interactions[J].Current Opinion in Biotechnology,1995,6(1):59-64
[45]Vidal M,Legrain P.Yeast forward and reverse 'n'-hybrid system[J].Nucleic Acid Res,1999,27(3):919-929
[46]Osman A.Yeast two-hybrid assay for studying protein-protein interactions[J].Methods Mol Biol,2004,270:403-422
[47]Fields S,Song O.A novel genetic system to detect protein-protein interactions[J].Nature,1989,340(6230):245-246
[48]Miller J,Stagljar I.Using the yeast two-hybrid system to identify interacting proteins[J|.Methods Mol Biol,2004,261(3):247-262
[49]Harper JW,Adami GR,Wei N,etal.The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1cyclin-dependent kinases[J].Cell,1993,75(4):805-816
[50]Howard AD,McAllister G,Scott DF,etal.Orphan G-protein-coupled receptors and natural ligand discovery[J].Trends Pharmacol,Sci,2001,22(1):132-140
[51]Christopoulos A.Allosteric binding sites on cell-suface receptors:novel targets for drug discovery[J].Nat Rev Drug Discov,2002,1(3):198-210
[52]张传福.禽流感病毒NS1蛋白功能的研究:(博士学位论文].北京:中国人民解放军军事医学科学院,2007
[53]卢伊娜,胡慧,朱维良,等.GPR120的研究进展[J].生命科学,2008,20(2):275-278
[54]Fredriksson R,Helgi B,Schioth,etal.Seven evolutionarily conserved human rhodopsin G protein-coupled receptors lacking close relatives[J].FEBS Lett,2003,554(3):381-388
[55]刘飞,张幼怡.G蛋白偶联受体激活的单分子研究进展[J].生命科学,2008,20(1):53-57
[56]Allen M.S,Lee S.W Inherited diseases involving G protein-coupled receptors[J].Annu.Rev.Med.2004,55(1):27-39
[57]卢伊娜,胡慧,朱维良,等.GPR120的研究进展[J].生命科学,2008,20(2):275-278
[58]刘永学,余少平.孤儿G蛋白偶联受体及其作为新药靶点的重要意义[J].中国药理学通报,2003,19(6):601-604
[59]李延峰,郭玉璞,池田修一,等.家族性淀粉样多发性神经病-家系病理及基因点突变[J].中华神经科杂志,2001,34(1):19-22
[60]李圣青,遆新宇,刘阿茹.大鼠急性肺血栓栓塞后血中DBP、RBP和TTR表达降低[J].基础医学与临床,2007,27(1):72-75
[61]葛晓冬,刘友生,王晓东.脂多糖结合蛋白家族研究进展[J].中华烧伤杂志,2004,20(2):125-128
[62]侯一峰,周艳春,毛宝龄,等.LBP对LPS激活巨噬细胞内P38信号通路的影响[J].中国病理生理杂志,2003,19(9):1202-1205
[63]Strohmeier GR,Walsh JH,Klings ES,etal.Lipopolysaccharide binding protein potentiates airway reacticity in a murine model of allergic asthma[J].J.Immunol,2001,166(3):2063-2070
[64]Schumann RR,Leong SR,Flaggs GW,etal.Structure and function fo lipopolysaccharude binding protein[J].Science,1990,249(4975):1429-1431
[65]张竹梅,边建超.乙醇代谢酶基因多态与肿瘤关系研究进展[J].中华医学遗传学杂志.2001,18(1):62-64
[66]周泽云.乙醇脱氢酶在各类肝损伤中的变化[J].中国实验诊断学.2007,11(3):403-404
[2]甘孟侯.禽流感[M].北京:北京农业大学出版社,1995
[3]乔传玲,于康震.禽流感病毒结构蛋白及其免疫原性[J].预防兽医学进展,2000,2(4):4-7
[4]陈化兰,丁康震,卢景良.禽流感病毒及其分子生物学研究进展[J].国外兽医学畜禽传染病,1997,17(1):3-7
[5]Li K S,Guan Y,Wang J,Smith G J,etal.Genesis of a highly pathogenic and potentially pandemic H5N1influenza virus in eastern Asia[J].Nature,2004,430(8):209-213
[6]Kuiken,T,Rimmelzwaan G,Baars M,etal.Avian H5N1 influenza in cats[J].Science,2004,306(10):241
[7]Puthavathana,P,Auewarakul P,Charoenying P C,etal.Molecular characterization of the complete genome of human i(?)fluenza H5N1 virus isolates from Thailand[J].J Gen.Virol,2005,86(2):(?)23-433
[8]Mongkol Uiprasertkul,Pilaipan Puthavathana,Kantima Sangsiriwut,etal.Influenza A H5N1 replication sites in humans[J].Emerg.Infect.Dis,2005,11(7):1036-1041
[9]To KF,Chan P K,Chan K F,etal.Pathology of fatal human infection associated with avian influenza A H5N1 virus[J].J Med.Virol,2001,63(3):242-246
[10]Govorkova,E A,Rehg J E,Krauss S,etal.Lethality to Ferrets of H5N1 influenza viruses isolated from humans and poultry in 2004[J].J Virol,2005,79(4):2191-2198
[11]金奇.医学分子病毒学(第一版)[M].北京:中国农业出版社,2002
[12]Matrosovich MN,Matrosovich TY,Gray T,etal.Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium[J].J Virol,2004,78(22):12665-7
[13]Gocnik M,Fislova T,Sladkova T,etal.Antibodies specific to the HA2 glycopolypeptide of influenza A virus haemagglutinin with fusion-inhibition activity contribute to the protection of mice against lethal infection[J].J Gen Virol.2007,88(3):951-955
[14]Shahidi M,Kheiri MT,Amini B O S,etal.Molecular and phylogenetic analysis of human influenza virus among Iranian patients in Shiraz[J].Iran.J Med Virol.2007,79(6):803-810
[15]Lin D,Lan J,Zhang Z.Structure and function of the NS1 protein of influenza A virus[J].Acta Biochim Biophys Sin.2007,39(3):155-162
[16]Bornholdt ZA,Prasad BV.X-ray structure of the virus NS1 effector domain[J].Nat Struct Mol Biol.2006 13(6):559-560
[17]Hale BG,Randall RE.PI3K signaling during influenza A virus infections[J].Biochem Soc Trans.2007,35(2):186-187
[18]Hatada E,Saito S,Fukuda R.Mutant influenza viruses with a defective NS1 protein cannot block the activation of PKR in infected cell[J].J Virol,1999,73(3):2425-2433
[19]Muster T,Rajtarova J,Sachet M,etal.Bergmann M.interferon resisitance promotes oncolysis by influenza virus NS1-deletion mutants[J].Int J Cancer.2004 110(1):15-21
[20]Wang XY,Li M,Zheng HY,etal.Influenza A virus NS1 protein Prevents Activation of NF-B and induction of alpha/beta interferon[J].J Virol,2000,74(24):11566-11573
[21]Skehel J J,Wiley D C.Receptor binding and membrane fusion in virus entry:the influenza hemagglutinin[J].Annu Rev Biochem,2000,69(7):531-569
[22]Nayak D P,Hui E K,Barman S.Assembly and budding of influenza virus[J].Virus Res,2004,106(2):147-165
[23]陶攀,潘兹书,吴建国.A型流感病毒RNA聚合酶及其对基因组复制和转录的调控作用[J].中国病毒学,2006,21(3):304-308
[24]Maite Huarte,Ana Falco(?)n,Yuri Nakaya,etal.Threonine 157 of Influenza Virus PA Polymerase Subunit Modulates RNA Replication in Infectious Viruses[J].J Virol,2003,77(10):6007-6013
[25]JJ Sanz-Ezquerro,S.de la Luna,J.Ort(?)n,etal.Individual expression of influenza virus PA protein induces degradation of coexpressed proteins[J].J.Virol,1995,69(2-3):2420-2426
[26]Hara K.,Shiota M,Kido H,etal.Influenza virus RNA polymerase PA subunit is a novel serine protease with Ser624 at the active site[J].Genes Cells,2001,6(2):87-97
[27]JJ Sanz-Ezquerro,J.F(?)rnandez-Santar(?)n,T Sierra,etal.The PA influenza virus polymerase subunit is a phosphorylated protein[J].J.Gen.Virol,1998,79:471-478
[28]Perales B,Sanz-Ezquerro,P Gastaminza,etal.The replication activity of influenza virus polymerase is linked to the capacity of the PA subunit to induce proteolysis[J].J.Virol,2000,74(3):1307-1312
[29]Sanz-Ezquerro,J.J.,T.Z(u|¨)rcher etal.The amino-terminal one-third of the influenza virus PA protein is responsible for the induction of proteolysis[J].J.Virol.1996,70(3):1905-1911
[30]Li ML,Rao P,Robert MK.The acive sites of the influnenza cap-dependent endonuclease are on different polymerase subunits[J].the EMBO 2001,20(8):2078-2086
[31]He X J,Zhou J,Mark Bartlam,etal.Crystal structure of the polymerase PA_C-PB1_N complex from an avian influenza H5N1 virus[J].nature Letters,2008 09
[32]Hooker L,Strong R,Admas R.A sensitive,signle-tube assay to measure the enzymatic activities of influenza RNA polymerase and other poly(A) polymerase:application to kinetic and inhibitor analysis [J].Nucleic Acides Res.2001,29(13):2691-2698
[33]Fechter P,Mingay L,Sharps J,etal.Tow aromatic residues in the PB2 subunit of influenza A RNA polymerase are curcial for cap binding[J].J Biol Chem,2003,278(22):20381-20388
[34]赵宏宇,赵育莹,曲章义,等.流感病毒RNA聚合酶亚基PA6片断的克隆及表达[J].国外医学免疫学分册,2004,27(6):362-364
[35]乔伟振,孟继鸿,董晨,等.HCV不同编码区重组基因的蛋白表达丰度分析[J].南京医科大学学报自然科学版,2008,28(6):722-725
[36]Mikulasova A,Vareckova E,and Fodor E.Transcription and replication of the influenza a virus genome[J].Acta Virol,2000,44(5):273-282
[37]Mei-Ling Li,Ping Rao and Robert M.Krug.The active sites of the infuenza cap-dependent endonuclease are on different polymerase subunits[J].The EMBO Journal 2001,20(8):2078-2086
[38]Imen R E,Amine S,Noureddine K,etal.A strategy for high-level expression of soluble and functional human interferon a as a GST-fusion protein in E.coli[J].Protein Engineering,Design & Selection.2007,20(4):201-209
[39]Imen R E,Amine Sadok,Noureddine Khalaf,etal.A strategy for high-level expression of soluble and functional human interferon a as a GST-fusion protein in E.coli[J].Protein Engineering,Design &Selection,2007,12(4):1-9
[40]谢荣辉,朱函坪,翁景清,等.流行性乙型脑炎病毒囊膜蛋白截片段的克隆及表达[J].中国计划免疫,2007,13(6):533-535
[41]Sandro De Falco,Maria Grazia Ruvoletto,Antonio Verdoliva.Cloning and Expression of a Novel Hepatitis B Virus-binding Protein from HepG2 Cells[J].the J of Bio Chemi,2007,276(39):36613-36623
[42]Siigur J,Samel M,Tonismagi K,etal.Biochemical characterization of Lebetase,a direct-acting fibrinolytic enzyme from Vipera Lebetina snake venom[J].Thromb Res,1998,90(1):39-49
[43]李佰良.功能蛋白质组学[J].生命的化学,1998,18(6):1-3
[44]Jeremy Lubana,Stephen P Goff.The yeast two-hybrid system for studying protein-protein interactions[J].Current Opinion in Biotechnology,1995,6(1):59-64
[45]Vidal M,Legrain P.Yeast forward and reverse 'n'-hybrid system[J].Nucleic Acid Res,1999,27(3):919-929
[46]Osman A.Yeast two-hybrid assay for studying protein-protein interactions[J].Methods Mol Biol,2004,270:403-422
[47]Fields S,Song O.A novel genetic system to detect protein-protein interactions[J].Nature,1989,340(6230):245-246
[48]Miller J,Stagljar I.Using the yeast two-hybrid system to identify interacting proteins[J|.Methods Mol Biol,2004,261(3):247-262
[49]Harper JW,Adami GR,Wei N,etal.The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1cyclin-dependent kinases[J].Cell,1993,75(4):805-816
[50]Howard AD,McAllister G,Scott DF,etal.Orphan G-protein-coupled receptors and natural ligand discovery[J].Trends Pharmacol,Sci,2001,22(1):132-140
[51]Christopoulos A.Allosteric binding sites on cell-suface receptors:novel targets for drug discovery[J].Nat Rev Drug Discov,2002,1(3):198-210
[52]张传福.禽流感病毒NS1蛋白功能的研究:(博士学位论文].北京:中国人民解放军军事医学科学院,2007
[53]卢伊娜,胡慧,朱维良,等.GPR120的研究进展[J].生命科学,2008,20(2):275-278
[54]Fredriksson R,Helgi B,Schioth,etal.Seven evolutionarily conserved human rhodopsin G protein-coupled receptors lacking close relatives[J].FEBS Lett,2003,554(3):381-388
[55]刘飞,张幼怡.G蛋白偶联受体激活的单分子研究进展[J].生命科学,2008,20(1):53-57
[56]Allen M.S,Lee S.W Inherited diseases involving G protein-coupled receptors[J].Annu.Rev.Med.2004,55(1):27-39
[57]卢伊娜,胡慧,朱维良,等.GPR120的研究进展[J].生命科学,2008,20(2):275-278
[58]刘永学,余少平.孤儿G蛋白偶联受体及其作为新药靶点的重要意义[J].中国药理学通报,2003,19(6):601-604
[59]李延峰,郭玉璞,池田修一,等.家族性淀粉样多发性神经病-家系病理及基因点突变[J].中华神经科杂志,2001,34(1):19-22
[60]李圣青,遆新宇,刘阿茹.大鼠急性肺血栓栓塞后血中DBP、RBP和TTR表达降低[J].基础医学与临床,2007,27(1):72-75
[61]葛晓冬,刘友生,王晓东.脂多糖结合蛋白家族研究进展[J].中华烧伤杂志,2004,20(2):125-128
[62]侯一峰,周艳春,毛宝龄,等.LBP对LPS激活巨噬细胞内P38信号通路的影响[J].中国病理生理杂志,2003,19(9):1202-1205
[63]Strohmeier GR,Walsh JH,Klings ES,etal.Lipopolysaccharide binding protein potentiates airway reacticity in a murine model of allergic asthma[J].J.Immunol,2001,166(3):2063-2070
[64]Schumann RR,Leong SR,Flaggs GW,etal.Structure and function fo lipopolysaccharude binding protein[J].Science,1990,249(4975):1429-1431
[65]张竹梅,边建超.乙醇代谢酶基因多态与肿瘤关系研究进展[J].中华医学遗传学杂志.2001,18(1):62-64
[66]周泽云.乙醇脱氢酶在各类肝损伤中的变化[J].中国实验诊断学.2007,11(3):403-404