草鱼（Ctenopharyngodon idellus）IAP基因克隆、表达及酵母双杂交系统cDNA文库构建
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摘要
细胞凋亡(Apoptosis)是指在基因控制下的细胞自主的有序性的死亡,亦称为程序性细胞死亡(programme cell death,PCD)。lAP为近年来发现的新一类细胞内凋亡抑制蛋白,在许多物种中广泛存在,是细胞凋亡分子调节机制中非常重要而且保守的蛋白家族。目前,已有两类杆状病毒、七类哺乳类、三类节肢动物和果蝇lAP被陆续发现。但是对于草鱼lAP的研究很少。根据RACE(rapid amplification of cDNA ends)PCR方法扩增出草鱼肝脏细胞凋亡抑制蛋白基因的全长cDNA,其全长为2093 bp,包含5’端非编码区25 bp,3’端非编码区124bp,开放阅读框1944 bp。草鱼细胞凋亡抑制蛋白的开放阅读框编码467个氨基酸。核苷酸序列分析表明,在N端无氨基酸残基组成的信号肽；与斑马鱼的同源性最好,其次是斑猫鲿；在系统进化上,与斑马鱼、斑猫鲿共聚为一支。用半定量RT-PCR分析正常及细菌诱导下草鱼凋亡抑制蛋白基因在不同组织中的表达分布。结果显示：正常情况下,细胞凋亡抑制蛋白基因在所取的7种组织中均有表达,其中脑、肠和肾表达最高,肝脏和心脏次之,鳃和肌肉最低,但差异不明显。用嗜水产气单胞菌人工感染24h后,所有的组织中的细胞凋亡抑制蛋白基因mRNA水平平均降低26%左右,以肝脏和肌肉较为明显；感染48h后,所有的组织中的细胞凋亡抑制蛋白基因mRNA水平平均升高4%左右,其中心、肝脏和鳃分别升高8%、16%和19%,这表明凋亡抑制蛋白可能参与了机体对嗜水气单胞菌感染的免疫应答。本文还构建了酵母双杂交系统所需的cDNA质粒文库,用于鉴定基因重组表达蛋白后蛋白-蛋白的相互关系,来了解未知基因的功能和分子机制。
Apoptosis means gene under the control of cell death in the initiative known as Programmed cell death(PCD).However inappropriate apoptosis, such as large num-ber apoptotic cells or apoptosis will not lead to the occurrence of diseases.IAP were diseovered in recent years for a new kind of cell apoptosis inhibitory protein, which is widely presents in manys pecies.Molecular mechanisms of apoptosis is an very im-portant and conservative protein family, two baculovirus IAP, seven m- amnlals IAP,.three Arthropod IAP and Drosophila IAP were discovered. But fews tudies grass carps IAP.
In this contex, the full-length cDNA of inhibitor of apoptosis protein was cloned using rapid amplification cDNA ends (RACE). The sequence analysis shows that the full-length of inhibitor of apoptosis protein cDNA from grass carp was 2093bp, which contained a 1944bp of open reading frame (ORF) encoding a inhibitor of apoptosis protein precursor of 648 amino acid, flanked by a 25 bp of 5'-untranslated region (UTR) and a 124 bp of 3'-UTR; the analysis show that the amino acid sequence may not contain a signal peptide, and has a high similarity to that of zebrafish, followed by lctalurus punctatus; In the evolutionary tree, the grass carp was clustered with zebrafish and lctalurus punctatus. The expression of inhibitor of apoptosis protein gene in different tissues from bacteria-induced or health grass carp were detected with by semi-quantitative RT-PCR. The results indicated that: IAP is expressed in abundance in the brain, intestines and kidney in normal tissues, and there was a little expression in liver, heart, and gill, and muscle with minimum expression, However, all organizations difference is not significant. After infected with bacterial Aeromonas hydrohila,the mRNA level of all the tissues,dropped averagely of about 26 percent in liver and muscles, and after infection 48h, the mRNA level in all tissues increased about 4 percent higher, the liver and gills increased 8 percent, respectively,16 percent and 19 percent. The results indicated that inhibitor of apoptosis protein may be involved in the immune response for Aeromonas hydrophila infection.
Construction of yeast two-hybrid system plasmid cDNA library for identification of gene expression of recombinant protein after the protein-protein inter- relationship, to understand the function of unknown genes and molecular mechanisms.
Apoptosis means gene under the control of cell death in the initiative known as Programmed cell death(PCD).However inappropriate apoptosis, such as large num-ber apoptotic cells or apoptosis will not lead to the occurrence of diseases.IAP were diseovered in recent years for a new kind of cell apoptosis inhibitory protein, which is widely presents in manys pecies.Molecular mechanisms of apoptosis is an very im-portant and conservative protein family, two baculovirus IAP, seven m- amnlals IAP,.three Arthropod IAP and Drosophila IAP were discovered. But fews tudies grass carps IAP.
In this contex, the full-length cDNA of inhibitor of apoptosis protein was cloned using rapid amplification cDNA ends (RACE). The sequence analysis shows that the full-length of inhibitor of apoptosis protein cDNA from grass carp was 2093bp, which contained a 1944bp of open reading frame (ORF) encoding a inhibitor of apoptosis protein precursor of 648 amino acid, flanked by a 25 bp of 5'-untranslated region (UTR) and a 124 bp of 3'-UTR; the analysis show that the amino acid sequence may not contain a signal peptide, and has a high similarity to that of zebrafish, followed by lctalurus punctatus; In the evolutionary tree, the grass carp was clustered with zebrafish and lctalurus punctatus. The expression of inhibitor of apoptosis protein gene in different tissues from bacteria-induced or health grass carp were detected with by semi-quantitative RT-PCR. The results indicated that: IAP is expressed in abundance in the brain, intestines and kidney in normal tissues, and there was a little expression in liver, heart, and gill, and muscle with minimum expression, However, all organizations difference is not significant. After infected with bacterial Aeromonas hydrohila,the mRNA level of all the tissues,dropped averagely of about 26 percent in liver and muscles, and after infection 48h, the mRNA level in all tissues increased about 4 percent higher, the liver and gills increased 8 percent, respectively,16 percent and 19 percent. The results indicated that inhibitor of apoptosis protein may be involved in the immune response for Aeromonas hydrophila infection.
Construction of yeast two-hybrid system plasmid cDNA library for identification of gene expression of recombinant protein after the protein-protein inter- relationship, to understand the function of unknown genes and molecular mechanisms.
引文
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[2]Majno G Joris I. Apoptosis, Oncosis and necrosis an overview of cell death [J]. Am J Pathol.1995,1469(1):3-15
[3]Jaattela M. Escaping cell death survival proteins in cancer [J]. Exp. Cell Res.1999 248:30-43
[4]KimEJ, ParkTH. Anti-apoptosisengineering [J]. Biotechnol. Bioprocesseng,2003 (8):76-82
[5]王春平龚丽娟.细胞凋亡抑制蛋白IAP家族.国外医学、生理、病理科学与临床分册[J],2001(21)：10-12
[6]Birnbaum M J, Clem R J, Miller L K. An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs [J].J Virol.1994 (68):2521-2528
[7]Deveraux Q. L. Reed J. C. IAP family proteins suppressors of apoptosis [J]. Gene Develop.1999 (13):239-252
[8]Young S. S. Liston P.Xuan J. Yet al. Genomic organization and physical map of the human inhibitors of apoptosis HIAPI and HIAP2[J].Mamm Genome.1999 (10):44-48
[9]Wang S L. Hawkins C J. Yoo S J. et al. The Drosophila Caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID[J]. Cell.1999 (98):45-63
[10]Riedl S. J. Renatus M. Schwarzenbacher R. et al. Structural basis for the inhibi-tion of Caspase-3 by XIAP[J]. Cell.2001(104):791-800
[11]Srinivasula S. M. Hegde R. Saleh A. et al. A conserved XIAPinteraction motif in Caspase9 and Smac/DIABLO regulates Caspase activity and apoptosis[J].Na-ture.2001 (410):112-116
[12]Mary C. Abraham and Shai Shaham. Death without Caspases, Caspases without death[J]. TRENDS in Cell Biology.2004(14):184-193
[13]Cecile Faure, Caroline Gouhier, Salomon Z. Langer and David Graham. Quan-tification of a ladrenoceptor Subtypes in Human Tissues by Competitive RT-PC R Analysis[J]. Biochemical and Biophysical Research Communications.1995 (213):935-943
[14]Jian-Jian Li, Frederick Domann, and Larry W. Oberley. The Use of RT PCR to Distinguish Between Plasmid MnSOD Transcripts and Endogenous MnSOD mRNA[J]. Biochemical and Biophysical Research Communications.1995(2): 610-618
[15]Shiozaki E. N. Chai J. RigottiD. J. et al. Mechanism of XIAP-mediated inhibition of Caspase-9[J]. Mol Cell.2003:11(2):519-527
[16]Uren A. G. along L. Pakusch M, et al. Survivin and the innercentromere protein INCENP show similar cell-cycle localization and gene knockout phenotype [J]. Curr Biol.2000,10(21):1319-1328
[17]HolcikM. GordonB. W. KornelukR. G. The internal ribosome entry sitemediated translation of antiapoptotic protein XIAP is modulated by the heterogeneous nuc- lear ribonucleoproteins C1 and C2[J]. Mol Cell Biol.2003 (23):280-288
[18]白剑英(综述),孟紫强(审校)细胞凋亡抑制蛋白的研究进展中国公共卫生[J].2003,19(6)：761～763
[19]Crook NE, Cletm RJ, Miller LK, et al, J Virol,1993,67:2168-2174
[20]都昌胡,徐军凋亡蛋白抑制剂家族研究进展[J].World Chin J Digestol 2005July 15；13(13)：1581～1589
[21]Wang HW, Sharp TV, Koumi A, et al. Characterization of an antiapoptotic glycoprotein encoded by Kapo si's sarcoma-associated herpesvirus which rese-mbles a spliced variant of hum an Survivin[J]. EMBO J,2002,21:2602-2615
[22]Liston P, Young SS Mackenzie AE, Korneluk RG. Life and death decisions the role of the IAPs in modulating programmed cell death[J]. Apotptosis 1997,2: 423-441
[23]Holcik M, Gibson H, Korneluk RG XIAP:apoptotic brake and promising thera-pe utic target [J]. Apoptosis 2001; 6:253-261
[24]Andersen MH, Reker S, Becker JC, thor Straten P. The melanoma inhibitor of apoptosis protein:a target for spontaneous cytotoxic T cell responses[J]. Ivest Dermatol 2004; 122:392-399
[25]Yamashita M, Nishio H, Harada Y, Matsuo M, Yamamoto T.Significant increa-se in the number of the SMN2 gene copies in an adult-onset Tyape Ⅲ spinal muscul-ar atrophy patient with homozygous deletion of NAIP gene[J]. Eur Neurol 2004,52:101-106
[26]Escuin D, Rosell R. The anti-apoptosis Surviving gene and its role in human cancer an overview[J]. Clin Lung Cancer 1999,1:138-143
[27]王梅谭升顺Survivin:IAP家族新成员国外医学遗传学分册[J]200225 (4)：201-203
[28]Young SS, Liston P, Xuan JY, et al. Genomic organization and physical map of the human inhibitors of apoptosis:HIAP1 and HIAP2 [J]. Mamm Genome, 1999,10(1):44-48
[29]Roy N, Mahadevan MS, Mclean M, et al. The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy [J] Cell,1995,80(1):167-178
[30]Ambrosini G, Adida C, Altier D. A noval anti-apoptosis gene, Survivin, expressed in cancer and lymphoma [J]. Nat Med,1997,3(8):917-921
[31]Kasof GM, Gomes BC. Livivin, a novel inhibitor of apoptosis protein family member[J]. J Biol Chem,2001,276(5):3238-3246
[32]Vucic D, Kaiser WJ, Miller LK, et al. Inhibitor of apoptosis proteins physically interact with and block apoptosis induced by Drosophila proteins HID and GRI M.Mol[J]. Cell BiolJ Biol Chem,1998,271:33915-33921
[33]Harvey AJ, Bidwai AP, Miller LK, et al. Doom,a Product of the Drosophila mod(mdg4)Gene,Induces Apoptosis and Binds to Baculovirus Inhibitor of Apo-ptosis Proteins[J]. Cell Death Differ,1997b,4:733-734
[34]Roy N, Deveraux QL, Takashashi R, et al. The c-IAP-1 and cIAP-2 proteins are direct inhibitors of specific caspases[J]. EMBOJ,1997,16:6914-6925
[35]Deveraux QL, Takahashi R, Salvesen GS, et al. X-linked IAP is a direct inhibitor of cell-death proteases[J].Nature,1997,388:300-303
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