禽流感病毒核蛋白基因的克隆、表达及其相互作用蛋白的筛选
|
摘要
核蛋白(Nucleoprotein)基因是禽流感病毒(AIV)基因组的第5片段。
在AIV复制早期，NP与AIV的RNA及多聚酶蛋白共同装配成RNP复合体，通过RNP复合体感染宿主细胞上的某些大分子来影响AIV的转录、复制、装配及转运功能，是AIV感染的重要因素。所以，寻找宿主细胞中与核蛋白密切相关的蛋白研究它们之间的相互作用关系，将对阐明AIV感染的分子机制和防制禽流感具有重大意义。本课题旨在通过制备NP多克隆抗体，利用特异性抗体作为蛋白质研究的有效工具进行NP体内表达和相互作用的研究；同时，利用CytoTrap酵母双杂交系统筛选与NP相互作用的蛋白，以探索流感病毒-宿主细胞相互作用关系。
首先，首先从感染了H5N1亚型禽流感病毒的MDCK细胞培养液中收获病毒并提取总RNA，克隆了NP基因全长cDNA片段。
其次，构建了原核表达质粒pTIG／H5NP，并在大肠杆菌中大量表达并纯化了NP蛋白，获得了抗原。
第三，皮下注射免疫家兔制备多抗血清，制备了抗体亲和层析柱亲和纯化抗体。
第四，构建了CytoTrap酵母双杂交pSos／NP诱饵表达质粒；Western blotting验证了融合蛋白hSos-NP能在cdc25H(a)酵母菌中稳定表达。
最后，利用CytoTrap酵母双杂交系统，将诱饵质粒pSos／NP和人肺细胞文库质粒共转化酵母细胞，从文库中“钓”出了与NP结合的两个阳性克隆。这两个蛋白分别是聚ADP核糖聚合酶家族成员8(PARP8)和锌指蛋白658(ZNF658)。
Nucleoprotein gene is the fifth segment 9f genome of avian influenza virues.
Forepart, when AIV replicate, the RNA segments are contained within the virues envelopein association with the nucleoprotein and three subunits of viral polymerase (PA, PB1, andPB2),which together form the ribonucleoprotein (RNP) complex responsible for RNAreplication and transcription.That is the most important factors for virues infection.Therefore, it makes sense to illuminate the molecule mechanism of virues infection and findthe way of preventive from AI by finding the other proteins associated with NP in hostcells, and studying the relation of each-interacting.
This task aims at preparing of NP antibody, then using the antibody to study the NPexpressing protein in cells and relation of mutual-interacting.At the same time, using theCytoTrap yeast two-hybrid system to fish NP-interacting protein. This study aim is to questfor relation of mutual-interacting about AIV-host cells.
First, the virues RNA was obtained from the supernatant of MDCK cell infected H5N1Subtype avian influenza virues by RT-PCR, and the complete cDNA strands of NP wascloned.
Second, prokaryotic expression vector pTIG/H5NP was constructed, then it wastransformed into E.coli BL21(DE3)plysS for highly expression; NP prokaryotic expressionproduction was purificated.
Third, the purification of NP protein was injected into rabbit,and serums was collected toprepare NP antibody; at the same time, NP antibody affinity columniation was prepared topurificate antibody.
Fourthly, the bait vector of NP (pSos/NP) was constructed, and fuse proteins hSos-NPexpressing in cdc25H (a) yeast stably was confirmed by Western blotting.
At last,the bait vector pSos/NP and lung cell library were comtransformed into cdc25H-(a) yeast,and two putative positive clones combined with NP were fished utilizing CytoTrapyeast two-hybrid system. The two positive clones are respective Homo sapiens poly (ADP-ribose) polymerase family, member 8 (PARPS) and Homo sapiens zinc finger protein 658(ZNF658).
在AIV复制早期，NP与AIV的RNA及多聚酶蛋白共同装配成RNP复合体，通过RNP复合体感染宿主细胞上的某些大分子来影响AIV的转录、复制、装配及转运功能，是AIV感染的重要因素。所以，寻找宿主细胞中与核蛋白密切相关的蛋白研究它们之间的相互作用关系，将对阐明AIV感染的分子机制和防制禽流感具有重大意义。本课题旨在通过制备NP多克隆抗体，利用特异性抗体作为蛋白质研究的有效工具进行NP体内表达和相互作用的研究；同时，利用CytoTrap酵母双杂交系统筛选与NP相互作用的蛋白，以探索流感病毒-宿主细胞相互作用关系。
首先，首先从感染了H5N1亚型禽流感病毒的MDCK细胞培养液中收获病毒并提取总RNA，克隆了NP基因全长cDNA片段。
其次，构建了原核表达质粒pTIG／H5NP，并在大肠杆菌中大量表达并纯化了NP蛋白，获得了抗原。
第三，皮下注射免疫家兔制备多抗血清，制备了抗体亲和层析柱亲和纯化抗体。
第四，构建了CytoTrap酵母双杂交pSos／NP诱饵表达质粒；Western blotting验证了融合蛋白hSos-NP能在cdc25H(a)酵母菌中稳定表达。
最后，利用CytoTrap酵母双杂交系统，将诱饵质粒pSos／NP和人肺细胞文库质粒共转化酵母细胞，从文库中“钓”出了与NP结合的两个阳性克隆。这两个蛋白分别是聚ADP核糖聚合酶家族成员8(PARP8)和锌指蛋白658(ZNF658)。
Nucleoprotein gene is the fifth segment 9f genome of avian influenza virues.
Forepart, when AIV replicate, the RNA segments are contained within the virues envelopein association with the nucleoprotein and three subunits of viral polymerase (PA, PB1, andPB2),which together form the ribonucleoprotein (RNP) complex responsible for RNAreplication and transcription.That is the most important factors for virues infection.Therefore, it makes sense to illuminate the molecule mechanism of virues infection and findthe way of preventive from AI by finding the other proteins associated with NP in hostcells, and studying the relation of each-interacting.
This task aims at preparing of NP antibody, then using the antibody to study the NPexpressing protein in cells and relation of mutual-interacting.At the same time, using theCytoTrap yeast two-hybrid system to fish NP-interacting protein. This study aim is to questfor relation of mutual-interacting about AIV-host cells.
First, the virues RNA was obtained from the supernatant of MDCK cell infected H5N1Subtype avian influenza virues by RT-PCR, and the complete cDNA strands of NP wascloned.
Second, prokaryotic expression vector pTIG/H5NP was constructed, then it wastransformed into E.coli BL21(DE3)plysS for highly expression; NP prokaryotic expressionproduction was purificated.
Third, the purification of NP protein was injected into rabbit,and serums was collected toprepare NP antibody; at the same time, NP antibody affinity columniation was prepared topurificate antibody.
Fourthly, the bait vector of NP (pSos/NP) was constructed, and fuse proteins hSos-NPexpressing in cdc25H (a) yeast stably was confirmed by Western blotting.
At last,the bait vector pSos/NP and lung cell library were comtransformed into cdc25H-(a) yeast,and two putative positive clones combined with NP were fished utilizing CytoTrapyeast two-hybrid system. The two positive clones are respective Homo sapiens poly (ADP-ribose) polymerase family, member 8 (PARPS) and Homo sapiens zinc finger protein 658(ZNF658).
引文
[1] 甘孟侯．禽流感[M]．北京：北京农业大学出版社，2002，13-78．
[2] 傅生芳，独军政，常惠芸，等．禽流感病毒的分子生物学研究进展[J]．动物医学进展，2005，26 (5)：22-24．
[3] 任淑敏，秦东．禽流感病原学研究现状及进展[J]．世界感染杂志，2004，4(4)：338-340．
[4] 付朝阳，邢大昌，唐秀英，等．高致病力禽流感的流行与防治研究进展[J]．中国预防兽医学报，2001，23(5)：393-396．
[5] 唐小龙，蔡淑玉，谢春梅等．禽流感病毒基因组研究[J]．热带病与寄生虫学，2005，3(3)：186-189．
[6] Sidhartha K. Biswas, Paul L. Influenza Virus Nucleoprotein Interacts with Influenza Virus Polymerase Proteins[J]. Virology, 1998, 8: 5493-5501.
[7] Shimizu K, Kuroda K. Expression of host genes in influenza virus infected cells[J]. Uirusu, 2004, Dec, 54(2): 189-196.
[8] Viseshakul N, Thanawongnuwech R, et al. The genome sequence analysis of H5N 1 avian influenza A virus isolated from the outbreak among poultry populations in Thailand [J]. Virology. 2004, Oct 25, 328(2): 169-76.
[9] Anna Walduck, Thomas Rudel and Thomas F Meyer. Proteomic and gene profiling approaches to study host responsesto bacterial infection[J]. Current Opinion in Microbiology, 2004, 7:33-38.
[10] Nakagawa, Y, N. Kimura, T. Toyoda, et al. The RNA polymerase PB2 subunit is not required for replication of influenza virus genome but is involved in capped mRNA synthesis[J]. Virol, 69:728-733.
[11] Scholtissek, Kimura. T. Molecular evolution of influenza viruses[J]. Virus Genes, 1995, 11 (223): 209-215.
[12] Webster R G, Kawaoka Y. Efficacy of nucleoprotein and haemagglutinin antigens expressed in fowlpox virus as vaccine for influenza in chickens[J]. Vaccine, 1991, 9 (5):303-309
[17] Fields S, Song O. A novel genetic system to detect protein-protein interactions[J]. Nature, 1989, 340 (20):245-246.
[18] Vidal M, Braclunann RK, Fataey A, et al. Reverse two-hybrid and one-hybrid systems to detect dissociation of protein and protein-DNA interactions[J]. Proc Nati Acid Sci USA, 1996, 93:10315-10320.
[19] Frederickson RM. Micromolecular match making advances in two-hybrid and related technologies[J]. CurrO pin Biotech, 1998, 9:90-98
[20] Brent R, Ptashne M. A bacterial repressor protein or a yeast transcriptional terminator can block upstream activation of a yeast gene[J]. Nature, 1984, 312(5995): 612
[21] Hope IA, Struhl K. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast[J]. Cell, 1986, 46(6): 885.
[22] Keegan IJ, Gill G, Ptashne M. Separation of DNA binding from the transcription activating function of a eukaryotic regulatory protein[J]. Science, 1986, 23 (4): 699.
[23] Stephens DJ, Banting G. The use of yeast two-hybrid screens in studies of protein: protein interactions involved in tracking[J]. Trafic, 2000, 9(101):763.
[24] 萨姆布鲁克J，费里奇EF，曼尼阿蒂斯T(金冬雁，黎孟枫，等译)．分子克隆实验指南[M]．北京：科学出版社，1992：846-854．
[25] Causier B. Studying the interactome with the yeast two-hybrid system and mass spectrometry[J]. Mass Spectrom Rev, 2004, 23(5): 350
[26] Aronheim A, Zandi E, Hennemann H, et al. Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions. Mol Cell Biol, 1997, 17:3094
[27] Jiang R, Carlson M. Glucose regulations protein interactions within the yeast SNFI protein kinase complex[J]. Genes Dev, 1996, 10: 3105.
[28] Huang W, Wang SL, Lozano G, de Crombrugghe B. cDNA library screening using the SOS recruitment system[J]. Biotechniques. 2001, 30(1): 94.
[29] 李建华；陈利玉等．酵母双杂交系统在病原生物学研究中的应用[J]．微生物学免疫学进展，2006，3：28-31
[30] Robert E. O'neill and Peter Palese. NPI-1, the Human Homolog of SRP-1, Interacts with Influenza Virus Nucleoprotein. [J]. Virology. 206, 116--125 (1995)
[31] Ping Wang, Peter Palese, and Robert E. O'neill The NPI-1/NPI-3 (Karyopherin a) Binding Site on the Influenza A Virus Nucleoprotein NP is a Nonconventional Nuclear Localization Signal [J]. Virology., 1998, 71 (3): 1850-1856
[32] 喻宏，姚立红，张智清等．应用酵母双杂交系统筛选与乙型流感病毒BM2相互作用的蛋白质[J]．中华实验和临床病毒学杂志，2005，(19)2：182-185．
[29] Sambrook J, Fritsch, E. F., Maniatis, T. 2001. Molecular Cloning: A Laboratory Manual 3rd.
[30] 哈洛E，莱思D(沈关心，龚非力，等译)．抗体技术实验指南[M]．北京：科学出版社，2002，49-50
[31] Spieker-Polet H, Sehupathi P, Yam PC, et al. Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas[J]. Proc Natl Acad Sci USA, 1995, 92 (20): 9348-9352.
[32] Li J, Sai T, Berger M, et al. Human antibodies for immunotherapy development generated via a human B cell hybridoma technology[J]. Proc Natl Acad SciUSA, 2006, 103 (10): 3557-3562.
[33] Reichert JM, Rosensweig CJ, Faden LB, et al. Monoclonal antibody successes in the clinic [J]. Nat Biotechnol, 2005, 23 (9): 1073-1078.
[34] Rohrbach P , Broders O , Toleikis L , et al . Therapeutic antibodies and antibody fusion proteins[J]. Biotechnol Genet Eng Rev , 2003 ,20: 1372163.
[35] Reichert JM. Therapeutic monoclonal antibodies : trends in development and approval in the US[J]. Curr Opin Mol Ther,2002,4(2) : 110-118.
[36] Wu AM , Senter PD. Arming antibodies : prospect s and challenges for immuno-conjugates [J]. Nat Biotechnol ,2005 ,23 (9) : 1137-1146.
[37] Trikha M , Yan L , Nakada MT. Monoclonal antibodies as therapeutics in oncology[J]. Curr Opin Biotechnol, 2002 ,13(6) :609-614.
[38] Adams GP , Weiner LM. Monoclonal antibody t herapy of cancer [J]. Nat Biotechnol , 2005 ,23 (9) :1147-1157.
[39] Karpas A , Dremucheva A , Czepulkowski BH. A human myeloma cell line suitable for t he generation of human monoclonal antibodies[J]. Proc Natl Acad Sci USA , 2001 ,98 (4): 1799-1804.
[40] Carson KL. Flexibility — the guiding principle for antibody manufact uring [J]. Nat Biotechnol , 2005,23(9): 1054-1058.
[41 ] Carter P , Smith L , Ryan MR. Identification and validation of cell surface antigens for antibody targeting in oncology[J]. Endocr Relat Cancer, 2004 , 11 (4) :659-687.
[42] Lu DL , Collison AM, Kowalski TJ . The patentability of antibodies in the United States[J]. Nat Biotechnol, 2005 ,23 (9): 1079-1080.
[43] Reichert JM, Dewitz MC. Anti2infective monoclonal antibodies : perils and promise of evelopment[J]. Nat Rev Drug Discov, 2006 ,5 (3) :1912195.
[44] Zhu Z, Dimitrov AS , Chakraborti S , et al. Development of human monoclonal antibodies against diseases caused by emerging and biodefense related viruses[J]. Expert Rev Anti Infect Ther, 2006,4 (1) :57266.
[43] Uetz P, Giot L, Cagney G, et al. A comprehensive analysis of protein-protein interactions in Saccharomycesce revisiae[J]. Nature, 2000,23 (6770):623-627.
[44] T Chiba T, Ozawa R, et al. A comprehensive two-hybrid analysis to explore the yeast protein interactome[J]. Proc Natl Acad SciU,2001,91 (8): 4569-4574.
[45] Von Mering C, Krause R, Sne B, et al. Comparative assessment of large scale data sets of protein-protein interactions[J]. Nature, 2002. 417(6887): 399-403
[46] W alhout A J, Temple G F, Brasch M A, et al. Gateway TM Recombination and Cloning: Application to the cloning of large numbers of open reading flames or 0RF eomes [J]. Methods Enzymol, 2000,328: 575-592
[47] JamesP ,HalladayJ , et al .Genomicli braries and a host strain designed for highly efficient two-hybrid selection in yeast[J]. Genetics, 1996, 144:1425-1436.
[48] BendixenC ,Gangtoff S ,Rothstein R ,et al .A yeast mating-selection scheme for d etection of protein-protein interactions[J]. Nucleic Acids Res, 1994, 22:1778-1779.
[49] Li JJ ,Herskowita I.Isolation of 0RC6,a component of the yeast origin recognition complex by a one-hybid system[J]. Science, 1993, 262:1870-1874.
[50] Licitra E J,Liu J.A three-hybrid system for detecting small ligand-protein rceptor interactions[J]. Proc Natl Acid Sci USA, 1996, 93:12817-12821
[51] Licitra E J,Liu J.A three-hybrid system for detecting small ligand-protein rceptor interactions[J]. Proc Natl Acid Sci USA, 1996, 93:12817-12821
[52] Aronheim A,Zandi E ,Hennemann H ,et al .Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions[J]. Mol Cell Biol, 1997, 17:3094-3102.
[54] Hubsman M,Yudkovsky G,Aronheim A. A novel for the identification of protein -protein interaction with integral membrane proteins[J]. Nucleic Acids Resesrch, 2001, 29:218
[55] Vidal M, Braun P, Chen E, et al. Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system[J]. Proc Natl Acid Sci USA, 1996, 93:10321-10326
[56] Chiu M, Katz H, Berln V. RAPTI. A mammalian homolog of yeast for interacts with the FKBP12/rapamycin complex[J]. Proc Natl Acid Sci USA, 1994,91: 12574-12578.
[57] RossiF ,CharltonC A,Blau HM.Monitoring protein-protein interactions in intact eukaryotic cells by a-galactosidase complementation[J]. Proc Natl Acid Sci USA, 1997, 94:8405-8410.
[57] Vidal M, Braclunann RK, Fataey A, et al. Reverse two-hybrid and one-hybrid systems to detect dissociation of protein and protein-DNA interactions[J]. Proc Natl Acid Sci USA, 1996, 93:10315-10320.
[58] Frederickson RM. Micromolecular match making advances in two-hybrid and related technologies[J]. CurrO pin Biotech , 1998, 9:90-98
[59] May N, Dubaele S, etal. Application to the high-throughput screening of small molecules as potential inhibitors Cell, 2004; 116(4): 541-550
[60] Mark R Cooson, Paul G. Ince, Philip A Usher, Pamela J Shaw. Poly (ADP-ribose)polymerase in found in both the nucleus and cytopham of human CNS neurons[J]. Brain Besearch, 1999, 834: 182-185.
[61] Hans-Rudolf Hotz, Susanne Bebinger, John Flaspohler, et al. PARP gene expression: control at many levels[J]. Molecular and Birmhemleal Parasitology, 1998, 91: 131-143.
[62] Karen L Gilliams-Francis, Aurora A Quaye, Janice R Naegele. PAPR cleavage DNA fragmenfion, and pyknosls during excitotoxln-induced neuronal death[J]. Experimental Neurology, 2003, 184: 359-372.
[63] Funan Huang, Mohan C, Vemuri J S Schneider. Modulation Of ATP levels alters the of hydrogen peroxide-induced cell death in primary cortical cultures: efects of putative neurpmtecdve agents [J]. Brain Research, 2004, 997: 79-88.
[64] Yoshihiro Higuchi. Chromosomal DNA fragmentation in apoptosis and necrosis indIIced by oxidative stress[J]. Biochemical Phannaeolngy, 2003, 66: 1527-1535.
[65] A Ivana Seovassa , Marc Die-rich . Modulation of poly(ADP-ribosylation) in apoptofic cells[J]. Biochemical Pharmacology, 2004. 1041-1047.
[67] Michael A, Moskowhz, Dean A Le, Michael J Whalen. Caspase and upstream mechanisms in central nervous system ischemic injury [J]. International Congress Series, 2003, 1252: 155-161.
[68] Ksrson S, Putt, Paul J Hergenmther. An enzymatic assay for poly (ADP-ribose) polymerase-1 (PARP-I) via the chemical quantition of NAD~+[J]. Analytical Biechemis, 2004, 326: 78-86.
[69] Southan GJ,Szabo C. Poly(ADP-ribose) polymerase inhlbitors[J]. Curt Med Chem, 2003, 10(4): 32-40.
[70] Okamoto H, Yamamoto H. DNA strand breaks and poly(ADP-ribose)synthetase activation in pancreatic islets-a new aspectto devdopment Of insulin—dependent diabetes and parterre B-cell tumors[J]. Prinoe∞Takn itsu srmp, 1993, 13: 297-308.
[71] Suarez-Pinzon WL, MableyJG, Power R. etal. Poly(ADP-ribose)polymerase inhibition prevents spontanous and recurrent autoimmune diabetes in NOD mice by inducing apoptosis of islet [J]. Diabetes, 2003, 52(7): 1683-1685.
[72] FurlinlG, Re MC. Increased Poly(ADP-ribose)polymerase activity in cells infected by human immunodefieiency vires type-l[J]. Microbiloglica, 1991, 14(2): 141-148.
[73] Smith S, Lange T. Tankyrae promotes telomere elongation in human cells[J]. Curt Biol, 2000, 10(20) : 299-302.
[74] Menissier Mureia J, Rieod M, Tarrier L, et al. Functional interaction between PARP-1 and PARP-2 in chromosome stability and embryonic development in mouse[J]. EMBO J, 2003,22(9): 55-63.
[75] Collins L Stone J R, Williams A J. All in the family: the BTB / POZ, KRAB, and SCAN domains[J]. Mol Cell Biol, 2001, 21(11): 3609-3615.
[76] Posttgo A , Sheppard A M, Musenski M L.c-Myb and Ets proteins synergize to overcome tran scriptional repression by ZEB[J].European Molecular Biology Organization of ournal, 1997, 16(13): 3 924-3 934.
[77] Posrgo A, Dean C. Expression and function of members of the znf-1 family of zinc finger homeodomain repressors. Proceeding National Academic Science USA, 2000, 12: 631-396.
[78] Brewsier R, Lee J, Ruizi Altaba A , et al.Gli/Zic factors pattern the neural plate by defining domains of cel diferentiation[J]. Nature, 1998, 393(6685): 579. 583.
[79] Rues W, Kimb W, Jund Y, et al. Nucleotide sequence and cell cycle-associated diferential expression of ZNF518, a novel Kruppel type zinc finger protein gene[J]. Biochim Biophys Acta, 2001, 1522: 230-237.
[80] Yemg J J. A novel zinc finger protein, ZZaPK, interacts with ZAK and stimulates the ZAK-expressing cels re-entering the cell cycle[J]. Biochem Biophys Res Commun, 2003, 301: 71-77.
[81]Ciani E, Frenqueel M, Contestabll A. Developmental expression of the cell cycle and apoptosis con- troling gene in the rat cerebellum and in cultures of cerebellar granule cells[J]. Brain Res Dev Brain Res, 2003, 142: 193-202.
[2] 傅生芳，独军政，常惠芸，等．禽流感病毒的分子生物学研究进展[J]．动物医学进展，2005，26 (5)：22-24．
[3] 任淑敏，秦东．禽流感病原学研究现状及进展[J]．世界感染杂志，2004，4(4)：338-340．
[4] 付朝阳，邢大昌，唐秀英，等．高致病力禽流感的流行与防治研究进展[J]．中国预防兽医学报，2001，23(5)：393-396．
[5] 唐小龙，蔡淑玉，谢春梅等．禽流感病毒基因组研究[J]．热带病与寄生虫学，2005，3(3)：186-189．
[6] Sidhartha K. Biswas, Paul L. Influenza Virus Nucleoprotein Interacts with Influenza Virus Polymerase Proteins[J]. Virology, 1998, 8: 5493-5501.
[7] Shimizu K, Kuroda K. Expression of host genes in influenza virus infected cells[J]. Uirusu, 2004, Dec, 54(2): 189-196.
[8] Viseshakul N, Thanawongnuwech R, et al. The genome sequence analysis of H5N 1 avian influenza A virus isolated from the outbreak among poultry populations in Thailand [J]. Virology. 2004, Oct 25, 328(2): 169-76.
[9] Anna Walduck, Thomas Rudel and Thomas F Meyer. Proteomic and gene profiling approaches to study host responsesto bacterial infection[J]. Current Opinion in Microbiology, 2004, 7:33-38.
[10] Nakagawa, Y, N. Kimura, T. Toyoda, et al. The RNA polymerase PB2 subunit is not required for replication of influenza virus genome but is involved in capped mRNA synthesis[J]. Virol, 69:728-733.
[11] Scholtissek, Kimura. T. Molecular evolution of influenza viruses[J]. Virus Genes, 1995, 11 (223): 209-215.
[12] Webster R G, Kawaoka Y. Efficacy of nucleoprotein and haemagglutinin antigens expressed in fowlpox virus as vaccine for influenza in chickens[J]. Vaccine, 1991, 9 (5):303-309
[17] Fields S, Song O. A novel genetic system to detect protein-protein interactions[J]. Nature, 1989, 340 (20):245-246.
[18] Vidal M, Braclunann RK, Fataey A, et al. Reverse two-hybrid and one-hybrid systems to detect dissociation of protein and protein-DNA interactions[J]. Proc Nati Acid Sci USA, 1996, 93:10315-10320.
[19] Frederickson RM. Micromolecular match making advances in two-hybrid and related technologies[J]. CurrO pin Biotech, 1998, 9:90-98
[20] Brent R, Ptashne M. A bacterial repressor protein or a yeast transcriptional terminator can block upstream activation of a yeast gene[J]. Nature, 1984, 312(5995): 612
[21] Hope IA, Struhl K. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast[J]. Cell, 1986, 46(6): 885.
[22] Keegan IJ, Gill G, Ptashne M. Separation of DNA binding from the transcription activating function of a eukaryotic regulatory protein[J]. Science, 1986, 23 (4): 699.
[23] Stephens DJ, Banting G. The use of yeast two-hybrid screens in studies of protein: protein interactions involved in tracking[J]. Trafic, 2000, 9(101):763.
[24] 萨姆布鲁克J，费里奇EF，曼尼阿蒂斯T(金冬雁，黎孟枫，等译)．分子克隆实验指南[M]．北京：科学出版社，1992：846-854．
[25] Causier B. Studying the interactome with the yeast two-hybrid system and mass spectrometry[J]. Mass Spectrom Rev, 2004, 23(5): 350
[26] Aronheim A, Zandi E, Hennemann H, et al. Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions. Mol Cell Biol, 1997, 17:3094
[27] Jiang R, Carlson M. Glucose regulations protein interactions within the yeast SNFI protein kinase complex[J]. Genes Dev, 1996, 10: 3105.
[28] Huang W, Wang SL, Lozano G, de Crombrugghe B. cDNA library screening using the SOS recruitment system[J]. Biotechniques. 2001, 30(1): 94.
[29] 李建华；陈利玉等．酵母双杂交系统在病原生物学研究中的应用[J]．微生物学免疫学进展，2006，3：28-31
[30] Robert E. O'neill and Peter Palese. NPI-1, the Human Homolog of SRP-1, Interacts with Influenza Virus Nucleoprotein. [J]. Virology. 206, 116--125 (1995)
[31] Ping Wang, Peter Palese, and Robert E. O'neill The NPI-1/NPI-3 (Karyopherin a) Binding Site on the Influenza A Virus Nucleoprotein NP is a Nonconventional Nuclear Localization Signal [J]. Virology., 1998, 71 (3): 1850-1856
[32] 喻宏，姚立红，张智清等．应用酵母双杂交系统筛选与乙型流感病毒BM2相互作用的蛋白质[J]．中华实验和临床病毒学杂志，2005，(19)2：182-185．
[29] Sambrook J, Fritsch, E. F., Maniatis, T. 2001. Molecular Cloning: A Laboratory Manual 3rd.
[30] 哈洛E，莱思D(沈关心，龚非力，等译)．抗体技术实验指南[M]．北京：科学出版社，2002，49-50
[31] Spieker-Polet H, Sehupathi P, Yam PC, et al. Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas[J]. Proc Natl Acad Sci USA, 1995, 92 (20): 9348-9352.
[32] Li J, Sai T, Berger M, et al. Human antibodies for immunotherapy development generated via a human B cell hybridoma technology[J]. Proc Natl Acad SciUSA, 2006, 103 (10): 3557-3562.
[33] Reichert JM, Rosensweig CJ, Faden LB, et al. Monoclonal antibody successes in the clinic [J]. Nat Biotechnol, 2005, 23 (9): 1073-1078.
[34] Rohrbach P , Broders O , Toleikis L , et al . Therapeutic antibodies and antibody fusion proteins[J]. Biotechnol Genet Eng Rev , 2003 ,20: 1372163.
[35] Reichert JM. Therapeutic monoclonal antibodies : trends in development and approval in the US[J]. Curr Opin Mol Ther,2002,4(2) : 110-118.
[36] Wu AM , Senter PD. Arming antibodies : prospect s and challenges for immuno-conjugates [J]. Nat Biotechnol ,2005 ,23 (9) : 1137-1146.
[37] Trikha M , Yan L , Nakada MT. Monoclonal antibodies as therapeutics in oncology[J]. Curr Opin Biotechnol, 2002 ,13(6) :609-614.
[38] Adams GP , Weiner LM. Monoclonal antibody t herapy of cancer [J]. Nat Biotechnol , 2005 ,23 (9) :1147-1157.
[39] Karpas A , Dremucheva A , Czepulkowski BH. A human myeloma cell line suitable for t he generation of human monoclonal antibodies[J]. Proc Natl Acad Sci USA , 2001 ,98 (4): 1799-1804.
[40] Carson KL. Flexibility — the guiding principle for antibody manufact uring [J]. Nat Biotechnol , 2005,23(9): 1054-1058.
[41 ] Carter P , Smith L , Ryan MR. Identification and validation of cell surface antigens for antibody targeting in oncology[J]. Endocr Relat Cancer, 2004 , 11 (4) :659-687.
[42] Lu DL , Collison AM, Kowalski TJ . The patentability of antibodies in the United States[J]. Nat Biotechnol, 2005 ,23 (9): 1079-1080.
[43] Reichert JM, Dewitz MC. Anti2infective monoclonal antibodies : perils and promise of evelopment[J]. Nat Rev Drug Discov, 2006 ,5 (3) :1912195.
[44] Zhu Z, Dimitrov AS , Chakraborti S , et al. Development of human monoclonal antibodies against diseases caused by emerging and biodefense related viruses[J]. Expert Rev Anti Infect Ther, 2006,4 (1) :57266.
[43] Uetz P, Giot L, Cagney G, et al. A comprehensive analysis of protein-protein interactions in Saccharomycesce revisiae[J]. Nature, 2000,23 (6770):623-627.
[44] T Chiba T, Ozawa R, et al. A comprehensive two-hybrid analysis to explore the yeast protein interactome[J]. Proc Natl Acad SciU,2001,91 (8): 4569-4574.
[45] Von Mering C, Krause R, Sne B, et al. Comparative assessment of large scale data sets of protein-protein interactions[J]. Nature, 2002. 417(6887): 399-403
[46] W alhout A J, Temple G F, Brasch M A, et al. Gateway TM Recombination and Cloning: Application to the cloning of large numbers of open reading flames or 0RF eomes [J]. Methods Enzymol, 2000,328: 575-592
[47] JamesP ,HalladayJ , et al .Genomicli braries and a host strain designed for highly efficient two-hybrid selection in yeast[J]. Genetics, 1996, 144:1425-1436.
[48] BendixenC ,Gangtoff S ,Rothstein R ,et al .A yeast mating-selection scheme for d etection of protein-protein interactions[J]. Nucleic Acids Res, 1994, 22:1778-1779.
[49] Li JJ ,Herskowita I.Isolation of 0RC6,a component of the yeast origin recognition complex by a one-hybid system[J]. Science, 1993, 262:1870-1874.
[50] Licitra E J,Liu J.A three-hybrid system for detecting small ligand-protein rceptor interactions[J]. Proc Natl Acid Sci USA, 1996, 93:12817-12821
[51] Licitra E J,Liu J.A three-hybrid system for detecting small ligand-protein rceptor interactions[J]. Proc Natl Acid Sci USA, 1996, 93:12817-12821
[52] Aronheim A,Zandi E ,Hennemann H ,et al .Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions[J]. Mol Cell Biol, 1997, 17:3094-3102.
[54] Hubsman M,Yudkovsky G,Aronheim A. A novel for the identification of protein -protein interaction with integral membrane proteins[J]. Nucleic Acids Resesrch, 2001, 29:218
[55] Vidal M, Braun P, Chen E, et al. Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system[J]. Proc Natl Acid Sci USA, 1996, 93:10321-10326
[56] Chiu M, Katz H, Berln V. RAPTI. A mammalian homolog of yeast for interacts with the FKBP12/rapamycin complex[J]. Proc Natl Acid Sci USA, 1994,91: 12574-12578.
[57] RossiF ,CharltonC A,Blau HM.Monitoring protein-protein interactions in intact eukaryotic cells by a-galactosidase complementation[J]. Proc Natl Acid Sci USA, 1997, 94:8405-8410.
[57] Vidal M, Braclunann RK, Fataey A, et al. Reverse two-hybrid and one-hybrid systems to detect dissociation of protein and protein-DNA interactions[J]. Proc Natl Acid Sci USA, 1996, 93:10315-10320.
[58] Frederickson RM. Micromolecular match making advances in two-hybrid and related technologies[J]. CurrO pin Biotech , 1998, 9:90-98
[59] May N, Dubaele S, etal. Application to the high-throughput screening of small molecules as potential inhibitors Cell, 2004; 116(4): 541-550
[60] Mark R Cooson, Paul G. Ince, Philip A Usher, Pamela J Shaw. Poly (ADP-ribose)polymerase in found in both the nucleus and cytopham of human CNS neurons[J]. Brain Besearch, 1999, 834: 182-185.
[61] Hans-Rudolf Hotz, Susanne Bebinger, John Flaspohler, et al. PARP gene expression: control at many levels[J]. Molecular and Birmhemleal Parasitology, 1998, 91: 131-143.
[62] Karen L Gilliams-Francis, Aurora A Quaye, Janice R Naegele. PAPR cleavage DNA fragmenfion, and pyknosls during excitotoxln-induced neuronal death[J]. Experimental Neurology, 2003, 184: 359-372.
[63] Funan Huang, Mohan C, Vemuri J S Schneider. Modulation Of ATP levels alters the of hydrogen peroxide-induced cell death in primary cortical cultures: efects of putative neurpmtecdve agents [J]. Brain Research, 2004, 997: 79-88.
[64] Yoshihiro Higuchi. Chromosomal DNA fragmentation in apoptosis and necrosis indIIced by oxidative stress[J]. Biochemical Phannaeolngy, 2003, 66: 1527-1535.
[65] A Ivana Seovassa , Marc Die-rich . Modulation of poly(ADP-ribosylation) in apoptofic cells[J]. Biochemical Pharmacology, 2004. 1041-1047.
[67] Michael A, Moskowhz, Dean A Le, Michael J Whalen. Caspase and upstream mechanisms in central nervous system ischemic injury [J]. International Congress Series, 2003, 1252: 155-161.
[68] Ksrson S, Putt, Paul J Hergenmther. An enzymatic assay for poly (ADP-ribose) polymerase-1 (PARP-I) via the chemical quantition of NAD~+[J]. Analytical Biechemis, 2004, 326: 78-86.
[69] Southan GJ,Szabo C. Poly(ADP-ribose) polymerase inhlbitors[J]. Curt Med Chem, 2003, 10(4): 32-40.
[70] Okamoto H, Yamamoto H. DNA strand breaks and poly(ADP-ribose)synthetase activation in pancreatic islets-a new aspectto devdopment Of insulin—dependent diabetes and parterre B-cell tumors[J]. Prinoe∞Takn itsu srmp, 1993, 13: 297-308.
[71] Suarez-Pinzon WL, MableyJG, Power R. etal. Poly(ADP-ribose)polymerase inhibition prevents spontanous and recurrent autoimmune diabetes in NOD mice by inducing apoptosis of islet [J]. Diabetes, 2003, 52(7): 1683-1685.
[72] FurlinlG, Re MC. Increased Poly(ADP-ribose)polymerase activity in cells infected by human immunodefieiency vires type-l[J]. Microbiloglica, 1991, 14(2): 141-148.
[73] Smith S, Lange T. Tankyrae promotes telomere elongation in human cells[J]. Curt Biol, 2000, 10(20) : 299-302.
[74] Menissier Mureia J, Rieod M, Tarrier L, et al. Functional interaction between PARP-1 and PARP-2 in chromosome stability and embryonic development in mouse[J]. EMBO J, 2003,22(9): 55-63.
[75] Collins L Stone J R, Williams A J. All in the family: the BTB / POZ, KRAB, and SCAN domains[J]. Mol Cell Biol, 2001, 21(11): 3609-3615.
[76] Posttgo A , Sheppard A M, Musenski M L.c-Myb and Ets proteins synergize to overcome tran scriptional repression by ZEB[J].European Molecular Biology Organization of ournal, 1997, 16(13): 3 924-3 934.
[77] Posrgo A, Dean C. Expression and function of members of the znf-1 family of zinc finger homeodomain repressors. Proceeding National Academic Science USA, 2000, 12: 631-396.
[78] Brewsier R, Lee J, Ruizi Altaba A , et al.Gli/Zic factors pattern the neural plate by defining domains of cel diferentiation[J]. Nature, 1998, 393(6685): 579. 583.
[79] Rues W, Kimb W, Jund Y, et al. Nucleotide sequence and cell cycle-associated diferential expression of ZNF518, a novel Kruppel type zinc finger protein gene[J]. Biochim Biophys Acta, 2001, 1522: 230-237.
[80] Yemg J J. A novel zinc finger protein, ZZaPK, interacts with ZAK and stimulates the ZAK-expressing cels re-entering the cell cycle[J]. Biochem Biophys Res Commun, 2003, 301: 71-77.
[81]Ciani E, Frenqueel M, Contestabll A. Developmental expression of the cell cycle and apoptosis con- troling gene in the rat cerebellum and in cultures of cerebellar granule cells[J]. Brain Res Dev Brain Res, 2003, 142: 193-202.