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抑制差减杂交(Suppression Subtractive Hybridization,SSH)是一种高效鉴定和分离克隆差异表达基因的新技术。目前,该技术在分子生物学研究的各个领域得到了广泛的应用。本对SSH的技术原理、差减cDNA库构建的技术流程、常见问题分析及优缺点等作了较全面的介绍,可为研究们提供参考。 相似文献
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本文对目前常用的一些基因克隆方法包括:抑制性差减杂交、差异显示PCR、DNA代表性差 异分析、cDNA微量列阵法(microarray)、外显子捕获法(exon capturation)、序列基因表达测(serial analysis of gene expression,SAGE)和图位克隆(mapbased cloning)等等作了简要的介绍;同时还介绍了从基因片段获得 其全长的3'RACE或5'RACE技术。可供相关研究人员参考。 相似文献
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Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1×106ml-1, the percentages of conidium germination and appressorium formation were (97.98±0.67)% and (97.88±0.45)%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 pg total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA (complementary DNA) library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection. 相似文献
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抑制性消减杂交技术（SSH）是一种高效分离差异表达基因的方法,可以在转录水平研究生物机体在不同生理时期、环境变化、疾病等条件下的基因表达差异,因其具有操作简便、特异性强、背景低、重复性好的特点,已在多个领域得以广泛应用。简要概述SSH技术在鱼类生长发育过程、外界生物因素诱导机制、非生物逆境胁迫下的基因表达中的应用、效果及其应用前景,以期为水产生物的基因调控研究提供参考。 相似文献
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Jin QC Dong HT Peng YL Chen BS Shao J Deng Y Dai CE Fang YQ Lou YC Li YZ Li DB 《Journal of Zhejiang University. Science. B》2007,8(2):88-97
Appressorium is an infection structure of the phytopathogenic fungus Magnaporthe grisea. Analysis of gene expression profiles ofappressorium development provides insight into the molecular basis of pathogenicity and control of this fungal plant disease. A cDNA array representing 2927 unique genes based on a large EST （expressed sequence tag） database ofM. grisea strain Y34 was constructed and used to profile the gene expression patterns at mycelium and appressorium maturation stages. Compared with mycelia, 55 up-regulated and 22 down-regulated genes were identified in mature appressoria. Among 77 genes, 16 genes showed no similarity to the genome sequences of M. grisea. A novel homologue of peptidyl-prolyl cis-trans isomerase was found to be expressed at low-level in mature appressoria of M. grisea. The results indicated that the genes such as pyruvate carboxylase, phospholipid metabolism-related protein and glyceraldehyde 3-phosphate dehydrogenase involved in gluconeogenesis, lipid metabolism and glycolysis, showed differential expression in mature appressoria. Furthermore, genes such as PTHll, beta subunit of G protein and SGTI involved in cell signalling, were expressed differentially in mature appressoria. Northern blot analysis was used to confirm the cDNA array results. 相似文献
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利用常规病毒分离方法，自死亡病人肺组织病料剖检样品中成功分离到了强致病性SARS-Coy肺组织毒。根据已发表的SARS-Coy Tor2株序列，设计并合成了38条覆盖全长基因组的PCR引物。应用RT-PCR技术从实验感染的Vero E6细胞上清中成功地扩增出了各相应的cDNA重叠片段，分别克隆至pGEM-T载体后，构建了SARS-Coy肺组织毒全基因组cDNA文库。SARS-Coy肺组织毒全基因组cDNA文库的构建为进一步研究SARS-Coy的分子生物学特性奠定了基础。 相似文献
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Evaluation of ST13 gene expression in colorectal cancer patients 总被引：2，自引：0，他引：2
DONG Qing-hua 《Journal of Zhejiang University. Science. B》2005,6(12):1170-1175
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Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B 总被引：3，自引：0，他引：3
Chen XH Chen Z Yao HP Chen F Zhu HH Zhou HJ 《Journal of Zhejiang University. Science. B》2005,6(4):288-294
INTRODUCTION Chronic hepatitis B virus (HBV) infection is aserious clinical problem because of its wide distribu-tion and possible adverse consequences, such as he-patic decompensation, cirrhosis and/or primary livercancer (PLC). The natural course of chronic HBVinfection is characterized by a series of hepatitic flaresor exacerbations and remissions (Ganem and Prince,2004). The severity, extent, duration and frequency ofhepatic histopathological changes in hepatitic flaresare d… 相似文献
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乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)的慢性感染,与慢性病毒性肝炎、肝硬化、肝细胞癌(HCC)的发病密切相关,其发病机理涉及到很多基因的共同参与.病毒基因的复制和表达受到肝细胞中蛋白质因子的调节,肝炎病毒基因编码的蛋白与肝细胞中的蛋白能够结合,肝炎病毒蛋白在肝细胞中的表达对于肝细胞的基因表达谱产生影响,也可能是病毒性肝炎、肝硬化、肝细胞癌发病机制的重要机制.酵母单杂交技术、酵母双杂交技术、基因芯片技术、抑制性消减杂交技术、噬菌体展示技术、蛋白质分离纯化与基因克隆化的反向遗传学技术等,在肝炎病毒基因调控、肝炎病毒蛋白结合蛋白的研究、肝炎病毒蛋白反式激活靶基因的研究中都有重要的应用,是促进慢性病毒性肝炎发病机理研究,探索病毒性肝炎新型治疗技术和治疗方法的有效途径. 相似文献
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[目的]新的大肠癌相关性抗原EID3的基因克隆及其诊断价值研究.[方法]利用大肠癌病人体内血清中所含的对肿瘤抗原产生的特异性抗体筛选睾丸组织cDNA噬菌体表达文库和大肠癌组织cDNA噬菌体表达文库(SEREX),并用RT-PCR技术研究EID3 mRNA在正常组织和大肠癌传代细胞表达.[结果]睾丸组织cDNA噬菌体表达文库筛选得到了可以诱导大肠癌病人抗体免疫应答的新抗原EID3基因(Gen-bank NM_001008394.1).它们定位于染色体19q13.2,EID3含1个外显子.通过RT-PCR分析发现,EID3基因在43例大肠癌传代细胞株中,39例阳性,阳性率为90.7%.在正常组织中,除睾丸组织外不表达或有极低水平转录.[结论]EID3 mRNA表达检测用于诊断大肠癌,可能具有高特异性和高敏感性的特点.EID3蛋白被首次发现在大肠癌病人中能够诱导机体的抗体免疫应答,为一个新的大肠癌相关性抗原分子.其功能可能与抑制细胞的恶性增殖相关,并可进一步研究其用于治疗和诊断大肠癌的可行性. 相似文献
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Qing-yi Cao Feng Chen Jie Li Shan-shan Wu Jing Wang Zhi Chen 《Journal of Zhejiang University. Science. B》2010,11(5):366-377
Objective:To explore the mechanisms of fulminant hepatitis(FH) in the early stages,and to determine the critical pathways in its initiation and progression.Methods:Twelve BALB/c mice were divided into four groups:one group left as negative control and sacrificed immediately after injection of phosphate-buffered saline(PBS),and another three groups with concanavalin A(Con A) administration sacrificed at 1,3,and 6 h after injection.Affymetrix GeneChip Mouse 430 2.0 Array was employed to evaluate the expressi... 相似文献
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INTRODUCTION 1,25-dihydroxyvitamin D3[1,25(OH)2D3], the biologically active metabolite of vitamin D3, is a secosteroid hormone that not only regulates bone and calcium/phosphate metabolism but also regu-lates a number of other biological activities, in-cluding modulation of the immune response via specific receptors expressed in antigen presenting cells (APC) and activated T cells. Recently, in-creasing evidence showed that the modulatory role of 1,25(OH)2D3 on T cell phenotype and … 相似文献
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RACE技术的研究进展及其应用 总被引：3，自引：0，他引：3
cDNA末端快速扩增(RACE)技术是一种基于PCR反应的技术,它的的发展极大地方便了克隆全长cDNA3′端和5′端的工作。本文综述了RACE技术的原理、局限性及改进方法,以及RACE技术在基因表达方面的研究进展和应用前景。 相似文献
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孙亮先 《泉州师范学院学报》2004,22(2):94-99
对GenBank中9215条来源于水稻(Oryza sativa L.ssp．Indica)胚乳cDNA库的3’EST进行了分析，获得20个淀粉合成相关基因的表达丰度信息，研究发现淀粉合成的5个关键酶的编码基因，ADPG焦磷酸化酶基因、ADP—葡萄糖淀粉合成酶、淀粉分支酶、淀粉去分支酶、蜡质基因的表达丰度极高，表明该时期水稻胚乳内淀粉合成反应非常强烈，研究发现在淀粉合成途径中存在功能相关的基因协同表达的现象，章结合所获得的基因表达信息对淀粉合成的分子机理进行了探讨。 相似文献
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A mature appressorium cDNA library of rice blast fungus, Magnaporthe grisea, was constructed in a λTriplEx2 vector by SMART?cDNA library containing 2.37×106 independent clones about 100% of which harbor foreign cDNA inserts with average size of 660 bp. Of 9 randomly 相似文献
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LU Jian-ping LIU Tong-bao YU Xiao-yun LIN Fu-cheng 《Journal of Zhejiang University. Science. B》2005,(2)
A mature appressorium cDNA library of rice blast fungus, Magnaporthe grisea, was constructed in a ?TriplEx2 vector by SMARTTM cDNA library containing 2.37?106 independent clones about 100% of which harbor foreign cDNA inserts with average size of 660 bp. Of 9 randomly selected clones, 2 expressed sequence tags (ESTs) sequences did not have homologous EST sequences of M. grisea in GenBank. The appressorium cDNA library is suitable for gene expression analysis and function analysis of the late stages of appressorium formation and the early stages of penetration of M. grisea. 相似文献