肝癌组织差异表达基因cDNA序列的筛选与鉴定

目的：筛选并鉴定肝癌组织特异表达基因。方法：通过菌落原位杂交技术筛选用抑制消减杂交法构建肝癌与癌旁肝组织差异表达基因消减cDNA文库，用PCR方法进一步筛选出有插入片段的阳性克隆，将阳性克隆进行DNA测序和同源性比较分析，用Northern印迹方法对新的cDNA序列进行初步鉴定。结果：从消减文库中随机挑取的100个白色克隆中筛选出13个阳性克隆，DNA测序获得11个不同的cDNA序列；同源性比较分析表明，6个cDNA片段与在基因高度同源，5个cDNA片段为新的序列。其长度大于300bp的3个新序列，Norther印迹证实它都来源于肝癌组织。结论：用抑制消减杂交方法构建的肝癌差异表达基因消减cDNA文库富含肝癌特异表达基因，经验证的3个新的cDNA序列可能为肝癌特异的基因序列。     

一个新的小鼠腭裂候选基因cDNA序列的克隆

目的 克隆并筛选与小鼠腭裂发生相关候选新基因。方法 利用聚合酶链反应改良扣除杂交技术，克隆正常组与腭裂组小鼠腭突组织中差异表达的基因，经反向点杂交鉴定后挑选阳性克隆并测序及同源性分析，Northern印迹杂交检测所获的新基因mRNA的全长。结果 经大规模测序后得4个新的表达序列标签，其中1个片段全长为809bp，Northern印迹杂交结果显示为该基因的eDNA全长。结论 获得一个新的与小鼠腭裂发生相关的候选基因的cDNA全长。     

大鼠小脑部分表达序列标签的鉴定

目的:为了获取大鼠小脑特异表达的cDNA序列,筛选新的EST基因片段,以便进一步获取有意义的cDNA全基因。方法:以大鼠大脑、脑干mRNA逆转录cDNA作为driver(驱动)cDNA,大鼠小脑mRNA逆转录cDNA作为tester(测试)cDNA,经消减杂交法,消除与大脑及脑干相同的cDNA,并富集低丰度基因,从而获得大鼠小脑特异表达基因片段,以及低表达基因片段,克隆制备成大鼠小脑特异表达cDNA文库。结果:共挑选出32个阳性克隆质粒,测序得到34个不同基因片段的序列。最后用反Northern杂交去除假阳性,筛选出8个有意义的差异表达cDNA基因片段。同时,将测序结果与Genbank进行同源性比较,发现13个新的EST序列,并申请获得基因序列号(AW288461-AW288474)。结论:抑制消减杂交法是一种筛选特异表达基因的有效方法。本结果可为研究脑功能的分子机制提供有益的资料。     

胃癌高表达cDNA片段W41的克隆及组织表达谱分析

目的：从人胃癌组织中克隆新的相关易感基因。方法：利用cDNA末端快速扩增PCR技术得到了扩增片段，将其克隆、核苷酸序列分析，并将序列在GenBank进行同源性比较。利用Northern杂交、多组织Northern及基因表达系列分析了所得基因片段的组织表达谱。结果：获得1条533bp带有poly(A)尾的cDNA片段，可见加尾信号AATAAA，与GenBank基因数据库同源比较，该序列未见与任何已知基因同源，登录GenBank(登录号为AF325202）。该序列在胃癌组织的表达强度高于对应正常组织，多组织Northern及基因表达系列分析表明该序列在多种肿瘤组织中高表达，在正常组织中表达减弱。结论：得到了1条与胃癌可能相关的新的cDNA序列。     

浸润性突眼相关基因/cDNA片段的筛选

筛选浸润性突眼相关基因／cDNA片段，为探究浸润性突眼发生的分子机制莫定基础。采用抑制消减杂交技术，以甲亢伴突眼患者甲状腺组织的cDNA为tester，甲亢不伴突眼患者甲状腺组织的cDNA为driver，经过两轮消减杂交和两轮PCR，获得二者之间的消减PCR产物，将产物插入至pCR2．1载体，转化感受态细胞E．coli DH5α，进行蓝白筛选。随机挑取48个白色菌落，经过Colony PCR，其中有1个转化子的扩增产物为双带，其余均为0．2～2kb之间的单一条带。将Colony PCR产物点样于同一尼龙膜上，一式两份，分别与地高辛标记的甲亢伴突眼和甲亢不伴突眼患者甲状腺组织的cDNA探针杂交。以与甲亢伴突眼患者甲状腺组织的cDNA探针有杂交信号，而与甲亢不伴突眼患者甲状腺组织的cDNA探针无杂交信号或信号明显弱的转化子为阳性克隆。筛选的阳性率为50％左右，阳性克隆中的插入片段即是与浸润性突眼相关的基因／cDNA片段。     

低压缺氧延迟预适应小鼠海马差异表达基因的筛选及鉴定研究

目的：探索低压缺氧延迟预适应小鼠海马差异表达基因。 方法： 将近交系Babl/c小鼠放入减压舱，模拟海拔 7 000 m高度减压2.5 h/d，连续3 d。第3次减压毕36 h后，提取海马总RNA，SMART PCR合成cDNA，经抑制消减杂交，建立消减cDNA文库。随机挑取文库单克隆，用反向Northern杂交，分别对来自于正、反向文库的452个和74个克隆进行了筛选。 结果： 用消减探针筛选时，217个基因片段的杂交信号在延迟预适应海马增加2倍以上，85个基因片段的杂交信号则降低66%以下。用未消减探针筛选时，135个基因片段的杂交信号在延迟预适应海马增加2倍以上，44个基因片段的杂交信号则降低66%以下。对部分基因片段测序显示，小鼠线粒体细胞色素C氧化酶亚单位1、线粒体NADH脱氢酶亚单位1和6、小鼠DSS1和克隆号为IMAGE:3582855的基因在延迟预适应海马表达增加。克隆号为IMAGE: 3593193的基因、与成年雄性小鼠嗅脑的一个cDNA高度相似的基因及与小鼠bladder RCB-0544 MBT-2 cDNA高度相似的基因在延迟预适应海马表达降低。 结论： 小鼠海马低压缺氧延迟预适应多种基因表达发生改变，它们可能是产生低压缺氧延迟预适应的重要机制之一。     

限制性酶切片段差异显示技术搜寻和克隆肝癌相关基因

目的:筛选新的肝癌相关基因并对其作用进行研究，探讨肝癌的发生机制，从而对肝癌的早期诊断和治疗提供新的思路。 方法:应用限制性酶切片段差异显示技术对比研究手术切除的新鲜的原发性肝细胞癌组织及其周围相对正常的肝组织，通过放射自显影显示结果并筛选差异条带。对差异表达基因序列进行克隆、测序和GenBank同源性比对，进一步分析差异表达的基因片段。 结果:筛选出18条表达有差异的条带，其中肝癌组织表达上调的基因有16条，肝癌组织低表达或缺失，正常组织高表达的基因有2条。二次PCR扩增出3条差异条带，进行克隆、鉴定和测序，并进行同源性比较。其中1条基因与已知基因无明显同源性，可能为新的基因；1条基因序列与编码人类核糖体蛋白基因的cDNA同源性较高（86%）；另有1条基因序列与编码P选择蛋白的基因高度同源（98%）。 结论:共克隆鉴定了3条肝癌相关基因，并初步探讨其功能，为进一步探讨肝癌的发生机理提供了理论依据。     

人类睾丸生精细胞凋亡相关基因TSARG3的克隆

目的：克隆人类睾丸生精细胞凋亡相关基因TSARG3。方法：从已获得的小鼠稳睾和正常睾丸对照中表达量有明显差异的表达序列标签片段（BE644537）入手，构建人同源表达序列标签重叠群，应用基因特异性引物和载体特异性引物，在睾丸cDNA文库的DN或进行巢式PCR扩增、测序，对测序结果进行生物信息学分析。结果：从睾丸cDNA文库中分离出人类睾丸凋亡相关基因的5’末端而获得全长cDNA,命名为TSARG3，GenBank登录号为AF419291（保密期为1年），同时应用相同方法克隆了该基因在小鼠中的同源基因，GenBank登录号为AF419292。结论：获得人类睾丸生精细胞凋亡相关基因TSARG3，该基因可能与人类睾丸生精细胞凋亡有关。     

高温致金黄地鼠神经管畸形的上调表达基因筛选及其鉴定

目的:筛选高温致金黄地鼠神经管畸形的上调表达基因,探讨其分子生物学机制。方法:通过抑制性消减杂交技术获得消减产物,转化大肠杆菌DH5α,构建高温致金黄地鼠胚胎神经管畸形的消减cDNA文库;联合蓝白斑和菌落PCR方法筛选含插入片段的阳性克隆,进行测序和同源性分析,并经Northern杂交技术检测基因的表达。结果:成功构建高温致金黄地鼠胚胎神经管畸形的消减cDNA文库,其中2个克隆插入片段的基因序列与小鼠磷酸甘油酸酯激酶(Pgk-1)同源。Northern杂交证实该基因在高温致畸胚胎神经管的表达较其在同龄正常胚胎神经管明显升高。结论:Pgk-1的上调表达可能与高温致神经管畸形密切相关。     

胃癌及其癌前病变相关新基因GP1的克隆

目的 比较胃癌、癌前病变和正常胃黏膜之间基因表达的差异,寻找与胃癌发生、发展相关的基因。方法 用荧光mRNA差异显示技术（FDD）分离差异表达的基因片段,进行PCR再扩增。将扩增的cDNA片段克隆后进行测序,测序结果提交GenBank,经BLAST软件检索进行同源性分析。差异条带经Northern杂交验证。应用SMART RACE（Rapid Amplication of cDNA Ends）技术扩增GP1的全长cDNA,并应用生物信息学技术预测该基因的生物学功能。结果 发现1个在胃癌及癌前病变组织中低表达的而且在GenBank数据库中未找到同源序列的cDNA片段,为新的基因片段,并获得GenBank登陆号CD454989。GP1的全长cDNA序列为1362bp,编码生物学功能未明的具有267氨基酸的蛋白质BAA91562.1。结论 发现了一个在胃癌、癌前病变及正常胃黏膜组织中差异表达的新基因,它可能参与了胃癌的发生、发展过程。     

cDNA代表性差异分析法分离鼻咽癌上皮细胞株HNE1表达差异cDNA序列的初步研究

目的寻找鼻咽癌中差异性表达基因，包括与鼻咽癌发病相关的候选抑瘤基因。方法应用ｃＤＮＡ代表性差异分析法（ＲＤＡ），分离原代培养的正常人鼻咽上皮细胞与鼻咽癌细胞株ＨＮＥ１中差异表达的ｃＤＮＡ序列，Ｓｏｕｔｈｅｒｎ杂交和Ｎｏｒｔｈｅｒｎ杂交被用来分析差异性表达产物的来源，最后，将这些序列克隆到ｐＧＥＭ－Ｔｅａｓｙ载体中，并用链终止法测序。结果在第４轮杂交及扩增反应后，获得４条差异性条带。Ｓｏｕｔｈｅｒｎ杂交及Ｎｏｒｔｈｅｒｎ杂交证明，这些差异性片段来自作为“检测”扩增子的正常人鼻咽上皮，在鼻咽癌细胞株ＨＮＥ１中不表达或表达降低。序列分析这些差异性片段的克隆，发现一些序列是与已知基因高度同源的基因，包括一些看家基因；另有一些基因则为新基因序列。结论鼻咽癌的发生是一个多基因参与的过程，所获得的差异性片段中，与之同源的一些已知基因具有抑瘤功能。     

应用抑制性消减杂交技术克隆PS1TP1的反式激活基因

目的 应用抑制性消减杂交（SSH）技术构建乙型肝炎病毒（HBV）前-S1蛋白（preS1）反式激活蛋白1（PS1TP1）的相关基因cDNA消减文库,克隆PS1TP1反式激活相关基因,以期发现PS1TP1蛋白反式激活作用的靶位点.方法 以PS1TP1表达质粒PcDNA3.1（-）-PS1TP1转染HepG2细胞,以空载体pcDNA3.1（-）为对照;提取mRNA并逆转录为cDNA,进行两次消减杂交及两次抑制性PCR,将产物与pGEM-Teasy载体连接,构建cDNA消减文库.结果 文库扩增后得到90个阳性克隆,随机挑选43个克隆测序,并进行同源性分析,获得12种编码基因,其中10个为已知功能基因,另外2个为未知功能序列.结论 成功构建PS1TP1反式激活的相关基因cDNA消减文库,为今后进一步分析、研究病毒蛋白的致病机制奠定基础.为进一步研究PS1TP1蛋白的功能及其在HBV感染中的分子生物学机制提供理论依据和研究方法.     

Analysis of immune system gene expression in small rheumatoid arthritis biopsies using a combination of subtractive hybridization and high-density cDNA arrays

Subtractive hybridization of cDNAs generated from synovial RNA which had been isolated from patients with rheumatoid arthritis (RA) or normal controls was used in conjunction with high-density array hybridization to identify genes of immunological interest. The method was designed to detect gene expression in small needle biopsy specimens by means of a prior amplification of nanogram amounts of total RNA to full-length cDNA using PCR. The latter was cut with Rsa I, ligated with adapters, hybridized with unmodified driver cDNA, and subjected to suppression subtraction PCR. Differentially expressed products were cloned into E. coli and picked into 384 well plates. Inserts were obtained by PCR across the multiple cloning site, and the products arrayed at high density on nylon filters. The subtracted cDNAs were also labelled by random priming for use as probes for library screening. The libraries chosen were the subtracted one described above and a set of 45,000 ESTs from the I.M. A.G.E consortium. Clones showing positive hybridization were identified by sequence analysis and homology searching. The results showed that the subtracted hybridization approach could identify many gene fragments expressed at different levels, the most abundant being immunoglobulins and HLA-DR. The expression profile was characteristic of macrophage, B cell and plasma cell infiltration with evidence of interferon induction. In addition, a significant number of sequences without matches in the nucleotide databases were obtained, this demonstrates the utility of the method in finding novel gene fragments for further characterisation as potential members of the immune system. Although RA was studied here, the technology is applicable to any disease process even in cases where amounts of tissue may be limited.     

Cloning of Ureaplasma urealyticum DNA sequences showing genetic homology with urease genes from gram-negative bacteria

DNA chromosomic sequences from Ureaplasma urealyticum were shown, by DNA hybridization, to possess homology with Providencia stuartii urease genes. The homologous ureaplasma DNA fragment was cloned in Escherichia coli and a DNA probe, designated IC61 probe was specific for only the urease-producing U. urealyticum among the Mollicutes. Genetic homology shown between the IC61 probe and urease genes from Campylobacter pylori, E. coli and P. stuartii suggests that (1) the cloned DNA fragment from U. urealyticum contains urease encoding sequences, and that (2) the urease genes possess conserved sequences among phylogenetically distant prokaryotes.     

Physical and genetic analysis of DNA regions encoding the immunoglobulin A proteases of different specificities produced by Haemophilus influenzae.

The structural gene for immunoglobulin A protease (iga) from Haemophilus influenzae serotype d was cloned in pBR322. The gene was used as a probe for Southern hybridization analysis of chromosomal DNA from the five other H. influenzae serotypes (a, b, c, e, and f). In most cases strains from a single serotype exhibited a distinct pattern of restriction fragment(s) homologous to the iga gene probe which was unique for that serotype. Serotype f strains were unique in that they gave two distinct patterns of homologous restriction fragments which correlated well with the production of two different protease types by members of this group. An iga mutant of H. influenzae serotype d was isolated by introducing a 4-base-pair insertion into the cloned iga gene and using the altered DNA for transformation of an H. influenzae recipient. The resulting iga- mutant produced no immunoglobulin A protease but was otherwise indistinguishable from its iga+ parent in growth characteristics. Transformation of mutant cells with chromosomal DNA isolated from either a serotype d or a serotype c strain gave rise to iga+ transformants. Those obtained with serotype d DNA produced a type 1 protease, whereas those obtained with serotype c DNA produced either a type 1 protease (characteristic of serotype d) or a type 2 protease (characteristic of serotype c). Southern analysis of the latter transformants, using the iga gene probe, indicated that the type 1 transformants had a serotype d pattern of restriction fragments whereas the type 2 transformants had either a serotype c or a novel pattern of restriction fragments. These results indicate that there is considerable homology between the iga genes of the various serotypes and that the homologous sequences identified with the serotype d probe are the immunoglobulin A protease-coding sequences in each case.     

Pregnancy-specific beta 1 glycoprotein mRNA is present in placental as well as non-placental tissues

Six human SP1 clones were isolated from a term placental cDNA library by immunological screening. All six cDNA clones cross-hybridized. However, at least two classes of cDNA could be distinguished, based on the presence or absence of an EcoRI site in the insert. Northern blot analysis of human term placental mRNA with all six cloned inserts demonstrated the presence of two mRNA species of 1.6 and 2.4 kb, respectively. The amino acid sequences of tryptic fragments of pure human SP1 protein were determined for confirmation of the identity of these cDNA clones. Using one of the cloned cDNA as probe, two and ten hybridizing clones were isolated from a human testicular cDNA library and a HeLa cell cDNA library, respectively. Southern blot analysis of these clones showed strong hybridization with the SP1 cDNA probe under high stringency, indicating the presence of highly homologous mRNA species in these tissues.     

人肺癌中OPB7-1 cDNA片段的克隆定位

目的：寻找肺腺癌发生及转移的相关基因，探讨肺癌发生发展的分子机理。方法：应用细胞培养、逆转录－PCR、RH基因定位及RNA原位杂交方法研究确定肺癌相关基因。结果：OPB7－1 cDNA片段，经RH基因定位方法定位于1p31-1p34。在正常人肺cDNA文库、低转移能力肺腺癌细胞系AGZY83－a cDNA中存在的片段大小一致，但序列中存在个别碱基的差别。OPB7－1 cDNA片段（974bp）与GenBank中已知序列同源性差，但与其有同源性的3个毗连群克隆均位于1号染色体上（1p31-1p34）。24例临床病例分析发现，该片段在肺癌组织中均有不同程度的表达，在正常对照组织中未见明显表达，且在有淋巴结转移病例的肺癌组织中表达程度呈明显增高的趋势。结论：提示OPB7－1可能为新的基因，与肺癌的发生、转移可能有一定的相关性。     

实验性脑脓肿组织中G蛋白信号系统相关基因的克隆

目的 筛选和克隆实验性脑脓肿的早期相关基因，研究脑组织对病原菌的免疫反应过程中所涉及的分子机制。方法通过脑组织内直接注射金黄色葡萄球菌获得大鼠脑脓肿，利用mRNA荧光差异显示PCR技术比较脑脓肿组脑组织中mRNA的表达与正常对照组、手术对照组之间的差异，获得的差异表达cDNA片段经克隆、测序及BLAST软件进行同源性比较分析，并采用Noahem杂交和RT-PCR进行鉴定。结果共获得25条差异表达的cDNA条带，经Noahem杂交和RT-PCR鉴定其中17条为阳性（阳性率为68％）。克隆和测序结果显示其中3个差异表达片段与G蛋白相关的细胞信号传导系统有关：片段G18-1代表的鸟苷二磷酸解离抑制因子3（GD13）、片段G20-1代表的交集素（ITSN）以及片段C2-1代表的Ras相关蛋白（Rap1）。结论在实验性脑脓肿早期GD13、ITSN和Rap1差异表达，推导G蛋白信号系统的激活可能与脑脓肿的发病相关。