正常肝组织与肝癌组织差异表达基因消减cDNA文库的构建

目的 构建人正常肝组织与肝癌组织差异表达基因的消减cDNA文库。方法 采用新近建立的抑制消减杂交技术,以癌旁正常肝组织及肝癌组织作为对比材料,分离肝癌组织中不表达或低表达基因的cDNA片段,将其与T载体进行T/A连接构建文库,将连接产物用电穿孔法转化大肠杆菌进行文库扩增后,随机挑取100个白色克隆进行酶切鉴定。结果 扩增消减cDNA文库获得4000余个白色阳性克隆,随机挑取的100个白色克隆进行酶切鉴定。结果 扩增消减cDNA文库获得4000余个白色阳性克隆,随机挑取的100个白色克隆经酶切后均有200-600bp的插入片段。结论 用SSH法及T/A克隆技术成功构建了正常肝组织与肝癌组织差异表达基因的消减cDNA文库,该文库的建立为进一步筛选、克隆肝癌组织中失活或低表达的新的抑癌基因奠定了基因。     

应用抑制性消减杂交技术筛选血小板衍生生长因子上调的大鼠肝星状细胞靶基因

目的应用抑制性消减杂交（SSH）技术构建血小板衍生生长因子（PDGF）-BB刺激的大鼠HSC上调基因的cDNA消减文库,从分子生物学角度阐明PDGF在肝纤维化中的作用机制。方法以PDGF-BB刺激的HSC作为实验组,以未用PDGF-BB刺激的HSC作为对照组,分别收获细胞提取总mRNA,并逆转录为cDNA。经RsaⅠ酶切后将实验组cDNA分成两组,分别与两种不同的接头衔接,再与对照组cDNA进行两次消减杂交及两次抑制性PCR扩增。将产物与pGEM-Teasy载体连接,构建cDNA消减文库,并转化大肠杆菌进行文库扩增,随机挑选克隆经PCR扩增后进行测序及生物信息学分析。结果成功构建了PDGF-BB刺激HSC差异表达基因的cDNA消减文库。文库扩增后得到102个阳性克隆,经菌落PCR分析,得到93个200～1000 bp的插入片段。挑取含有插入片段的31个克隆进行测序,通过生物信息学分析获得13种已知基因序列,主要包括电压依赖性阴离子通道、热休克蛋白质47、Ras家族基因RAN等蛋白质基因。结论PDGF- BB作为最有效的促有丝分裂原在激活HSC过程中上调某些与细胞生长相关的蛋白质、参与细胞内代谢的蛋白质及分子伴侣蛋白质等基因的表达,这将为进一步阐明PDGF-BB刺激HSC介导肝纤维化的分子生物学机制提供理论依据。     

人大肠癌和癌旁组织消减cDNA文库的构建和鉴定

背景：抑制消减杂交(SSH)是一种较成熟的分离差异表达基因的方法，但用该方法构建大肠癌特异性基因消减cDNA文库和筛选相关基因的研究较少。目的：构建人大肠癌和癌旁组织的消减cDNA文库。方法：应用SSH技术分离大肠癌和癌旁正常组织差异表达基因的cDNA片段，将其与pGEM-T Easy载体连接，构建消减cDNA文库。将连接产物转化大肠杆菌DH5α进行文库扩增。随机挑取200个白色克隆，以聚合酶链反应(PCR)进行鉴定。结果：进行PCR扩增的200个克隆中，176个克隆有插入片段，片段分布于200～700bp。结论：成功构建了人大肠癌组织和癌旁组织差异表达基因的消减cDNA文库，为高通量筛选、克隆大肠癌特异性基因奠定了基础。     

人胰腺癌抑制消减cDNA文库的构建

目的应用抑制消减杂交技术(SSH)构建胰腺癌和正常胰腺组织间差异表达的抑制消减cDNA文库。方法分别提取胰腺癌(tester)和癌旁正常胰腺组织(driver)中的总RNA和mRNA．合成双链cDNA，经RsaI酶切后，将胰腺癌双链cDNA分为两组，分别加上不同的接头，再与正常胰腺组织cDNA进行两次消减杂交及两次抑制性PCR，分离出胰腺癌差异表达基因的cDNA片段。将该差异表达片段克隆至T／A载体，并转化大肠杆菌TOP10F’，经蓝白斑筛选后，再用PcR方法筛选阳性克隆，从而构建胰腺癌抑制消减cDNA文库。结果文库扩增后得到257个白色克隆，随机挑取50个阳性克隆进行PCR扩增分析，其中47个克隆有插入片段．克隆阳性率为94％，片段大小主要集中在300～600bp之间。结论成功构建了人胰腺癌抑制消减cDNA文库，为进一步筛选、克隆胰腺癌特异性表达基因奠定了基础。     

人胰腺癌抑制消减cDNA文库的构建

目的应用抑制消减杂交技术(SSH)构建胰腺癌和正常胰腺组织间差异表达的抑制消减cDNA文库.方法分别提取胰腺癌(tester)和癌旁正常胰腺组织(driver)中的总RNA和mRNA,合成双链cDNA,经Rsa Ⅰ酶切后,将胰腺癌双链cDNA分为两组,分别加上不同的接头,再与正常胰腺组织cDNA进行两次消减杂交及两次抑制性PCR,分离出胰腺癌差异表达基因的cDNA片段.将该差异表达片段克隆至T/A载体,并转化大肠杆菌TOP 10F',经蓝白斑筛选后,再用PCR方法筛选阳性克隆,从而构建胰腺癌抑制消减cDNA文库.结果文库扩增后得到257个白色克隆,随机挑取50个阳性克隆进行PCR扩增分析,其中47个克隆有插入片段,克隆阳性率为94%,片段大小主要集中在300～600bp之间.结论成功构建了人胰腺癌抑制消减cDNA文库,为进一步筛选、克隆胰腺癌特异性表达基因奠定了基础.     

应用抑制消减杂交技术筛选大肠癌新相关基因

目的利用抑制消减杂交技术（Suppression Subtractive Hybridization，SSH）构建人大肠癌差异表达cDNA文序，分离并克隆出大肠癌发病的相关新基因。方法运用抑制消减杂交技术分离大肠癌组织及癌旁正常黏膜差异表达基因的cDNA片断，将其与T载体连接构建文库，从库中随机挑取200个白色菌落．使用PCR方法鉴定阳性克隆并对其中50个克隆进行测序，将测序结果登录GenBank进行卜司源性对比分析，并对部分有意义的差异表达片断进行Virtual Northern Blot分析。结果成功构建人源性大肠癌消减cDNA文库，通过筛选获得20个差异表达的基因，其中有2个片断未检测到同源性序列，根据杂交结果考虑可能是存大肠肿瘤中差异表达的新基因。结论运用SSH技术成功构建了人大肠癌的差异表达的cDNA消减杂交文库，并筛选出2个存大肠肿瘤中差异表达的新基因。     

应用抑制性消减杂交技术筛选丙型肝炎病毒E1蛋白反式激活基因

目的　应用抑制性消减杂交 (SSH)技术构建丙型肝炎病毒 (HCV)E1蛋白反式激活基因差异表达的cDNA消减文库 ,克隆HCVE1蛋白反式激活相关基因。方法　以HCVE1表达质粒pcDNA3 .1( -) E1转染肝母细胞瘤细胞系HepG2细胞 ,以空载体pcDNA3 .1( -)为对照 ;制备转染后的细胞裂解液 ,从中提取mRNA并逆转录为cDNA ,经RsaI酶切后将实验组cDNA分成 2组 ,分别与 2种不同的接头衔接 ,再与对照组cDNA进行 2次消减杂交及 2次抑制性PCR ,将产物与T/A载体连接 ,构建cDNA消减文库 ,并转染大肠杆菌进行文库扩增 ,随机挑选克隆PCR扩增后进行测序及同源性分析。结果　成功构建人HCVE1蛋白反式激活基因差异表达的cDNA消减文库。文库扩增后得到 89个阳性克隆 ,进行菌落PCR分析 ,均得到 10 0 10 0 0bp插入片段。挑取 46个含有插入片段的阳性克隆测序分析 ,获得 44个已知基因序列和 2个未知基因。通过生物信息学分析获得其全长序列 ,已被GenBank收录。结论　应用SSH技术成功构建了HCVE1反式激活基因差异表达的cDNA消减文库。该文库的建立为进一步阐明HCVE1反式调节的靶基因及致肝脏疾病发生的分子生物学机制提供理论依据     

胰腺癌抑制消减cDNA文库的构建及质量分析

目的构建人胰腺癌抑制消减cDNA文库.方法从来源于同一标本的胰腺癌和癌旁正常组织分离poly(A)+ RNA,经反转录后,利用抑制消减杂交(SSH)方法,通过两轮杂交和两次抑制PCR构建了两种组织间差异表达基因的cDNA消减文库.结果挑取500个克隆进行PCR扩增检测是否有插入片段,结果显示其中 457个克隆有插入片段,片段大小范围为 250～750 pb.结论应用消减杂交的方法,再经适当的改进,在去除相同遗传背景的条件下,可以建立较特异的胰腺癌cDNA消减文库,该文库为进一步批量筛选胰腺癌发生的相关基因群并克隆胰腺癌相关表达基因,研究其胰腺癌发生的分子机理与生物学特性间的关系奠定了基础.     

应用抑制性消减杂交技术克隆乙型肝炎病毒全S蛋白反式激活基因

目的应用抑制性消减杂交(SSH)技术构建乙型肝炎病毒(HBV)全S蛋白反式激活基因差异表达的cDNA消减文库,克隆HBV全S蛋白反式激活相关基因.方法以HBV全S表达质粒pcDNA3.1(-)-全S转染HepG2细胞,以空载体pcDNA3.1(-)为对照;制备转染后的细胞裂解液,从中提取mRNA并逆转录为cDNA,经RsaI酶切后将实验组cDNA分成两组,分别与两种不同的接头衔接,再与对照组cDNA进行两次消减杂交及两次抑制性PCR,将产物与T/A载体连接,构建cDNA消减文库,并转染大肠杆菌进行文库扩增,随机挑选克隆PCR扩增后进行测序及同源性分析.结果成功构建人HBV全S蛋白反式激活基因差异表达的cD-NA消减文库.文库扩增后得到86个白色克隆,进行菌落PCR分析,均得到100-1000 bp插入片段.挑取35个含有插入片段的阳性克隆测序分析,获得33个已知基因序列,和2个未知基因,通过生物信息学分析获得其全长序列,其中之一命名为全S蛋白反式激活基因1(CSTP1),已在GenBank中注册,注册号:AY553877.未知基因的功能还正在研究中.结论应用SSH技术成功构建了HBV全S反式激活基因差异表达的cDNA消减文库.该文库的建立为进一步阐明HBV全S反式调节的靶基因及致肝病发生的分子生物学机制提供理论依据.     

乙型肝炎病毒X蛋白反式激活基因克隆化的研究

目的 应用抑制性消减杂交（SSH）技术构建乙型肝炎病毒X蛋白（HBX）反式激活基因差异表达的cDNA消减文库，克隆HBX反式激活相关基因。方法 以HBX表达质粒pcDNA3.1(-)-X转染HepG2细胞，以空载体pcDNA3.1(-)转染的HepG2细胞为对照。制备转染后的细胞裂解液，提取mRNA并逆转录为cDNA，经RsaⅠ酶切后，将实验组cDNA分成两组。分别与两组不同的接头衔接，再对照组cDNA进行两次消减杂交及两次抑制聚合酶链反应（PCR），将产物与T/A载体连接，构建cDNA消减文库，并转染大肠杆菌进行文库扩增，随机挑选克隆PCR扩增后进行测序及同源性分析。结果 成功构建人HBX反式激活基因差异表达的cDNA消减文库；文库扩增后得到85个白色克隆，进行菌落PCR分析。均得以200-1000bp插入片段，挑取含有插入片段的65个克隆进行测序，并通过生物信息学分析获得19种已知基因序列。和15个未知基因。结论 应用SSH技术成功构建了HBX反式激活基因差异表达的cDNA消减文库，该文库的建立为进一步阐明HBX反式调节的靶基因及致肝细胞癌发生的分子生物学机制提供理论依据。     

Construction and selection of subtracted cDNA library of mouse hepatocarcinoma cell lines with different lymphatic metastasis potential

AIM: In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential.METHODS: cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver, cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method. The isolated cDNA was cloned into T/A cloning vector. The ligation products were transformed into DH5 α competent cells. Individual dories were randomly selected and used for PCR amplification.Vector DNA from positive clones was isolated for sequencing.RESULTS: There were 800 positive clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts. Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes(mouse heat shock protein 84 ku, DNA helicase, ribosomal protein S13 ,ethanol induced 6 gene) and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent novel genes.CONCLUSION: A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques. The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential.     

Identification of differentially expressed genes in mouse hepatocarcinoma ascites cell line with low potential of lymphogenous metastasis

INTRODUCTION Tumor metastasis is an incident involving multiple genes. However, the number of metastasis related genes available nowadays is very limited to elucidate the puzzling process of metastasis. Therefore, more attentions have been paid to screen …     

日本血吸虫毛蚴侵袭前后湖北钉螺差异表达cDNA文库的构建与分析

目的构建湖北钉螺被日本血吸虫毛蚴侵袭前、后差异表达cDNA文库,筛选湖北钉螺免疫相关分子,为探讨病原与中间宿主的相互作用奠定基础。方法提取被日本血吸虫毛蚴侵袭前、后湖北钉螺的头足部组织总RNA,纯化mR-NA,反转录合成cDNA。分别以被日本血吸虫毛蚴侵袭前湖北钉螺(未处理组)和被日本血吸虫毛蚴侵袭后湖北钉螺(处理组)的头足部组织作为检测方(Tester)和驱动方(Driver),利用PCR方法选择cDNA杂交试剂盒分别进行正向和反向抑制性消减杂交。将获得的正向抑制性消减杂交产物克隆入pGEM-T载体,重组质粒转入E.coliDH5α,对菌液用PCR法扩增鉴定其中的插入片段。随机抽取354个阳性克隆进行DNA序列分析,将所得表达序列标签(ESTs)序列在线进行BLAST分析。结果从正向文库随机挑取的354个阳性克隆中测得350个ESTs序列,生物信息学分析发现了34个湖北钉螺新基因,其中1个与已知基因部分同源。结论成功建立了被日本血吸虫侵袭前、后湖北钉螺差异表达片段cDNA消减文库,发现了湖北钉螺新基因,为筛选与湖北钉螺天然免疫相关的分子、进一步探讨中间宿主与病原的相互作用及筛选血吸虫病传播阻断疫苗候选分子奠定了基础。     

Construction of a metastasis-associated gene subtracted cDNA library of human colorectal carcinoma by suppression subtraction hybridization

AIM: To construct a differentially-expressed gene subtracted cDNA library from two colorectal carcinoma (CRC) cell lines with different metastatic phenotypes by suppression subtractive hybridization. METHODS: Two cell lines of human CRC from the same patient were used. SW620 cell line showing highly metastatic potential was regarded as tester in the forward subtractive hybridization, while SW480 cell line with lowly metastatic potential was treated as tester in the reverse hybridization. Suppression subtractive hybridization (SSH) was employed to obtain cDNA fragments of differentially expressed genes for the metastasis of CRC. These fragments were ligated with T vectors, screened through the blue-white screening system to establish cDNA library. RESULTS: After the blue-white screening, 235 white clones were picked out from the positive-going hybridization and 232 from the reverse. PCR results showed that 200-700 bp inserts were seen in 98% and 91% clones from the forward and reverse hybridizations, respectively. CONCLUSIONS: A subtractive cDNA library of differentially expressed genes specific for metastasis of CRC can be constructed with SSH and T/A cloning techniques.     

Construction of a metastasis-associated gene subtracted cDNA library of human colorectal carcinoma by suppression subtraction hybridization

AIM:To construct a differentially-expressed gene subtractedcDNA library from two colorectal carcinoma (CRC) cell lineswith different metastatic phenotypes by suppressionsubtractive hybridization.METHODS:Two cell lines of human CRC from the samepatient were used.SW620 cell line showing highlymetastatic potential was regarded as tester in the forwardsubtractive hybridization,while SW480 cell line with lowlymetastatic potential was treated as tester in the reversehybridization.Suppression subtractive hybridization (SSH)was employed to obtain cDNA fragments of differentiallyexpressed genes for the metastasis of CRC.These fragmentswere ligated with T vectors,screened through the blue-white screening system to establish cDNA library.RESULTS:After the blue-white screening,235 white cloneswere picked out from the positive-going hybridization and232 from the reverse.PCR results showed that 200-700 bpinserts were seen in 98% and 91% clones from the forwardand reverse hybridizations,respectively.CONCLUSIONS:A subtractive cDNA library of differentiallyexpressed genes specific for metastasis of CRC can beconstructed with SSH and T/A cloning techniques.     

Screening differentially expressed genes in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis

AIM: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastatic Hca-F and its synogenetic cell line Hca-P with a low metastatic potential was constructed by suppression subtracted hybridization(SSH) method. The screened clones of the subtracted library were sequenced and GeneBank homology search was performed. RESULTS: Fourteen differentially expressed cDNA fragments of Hca-F were obtained with two novel genes. CONCLUSION: SSH is a useful technique to detect differentially expressioned genes and an effective method to clone novel genes.     

Identification of differentially expressed genes in normal mucosa, adenoma and adenocarcinoma of colon by SSH

Abbreviation CRC colorectal cancer.SSHsuppression subtrsctive hybridization.LD PCR longdistance polymerase chain reaction.HLA human leukocyteantigen.IGRBP insulin-like growth factor binding protein.GN guanylin,EF1 elongation factor 1AIM To construct subtracted cDNA libraries and furtheridentify differentially expressed genes that are related tothe development of colorectal carcinoma(CRC).METHODS Suppression subtractive hybridization(SSH)was done on cDNAs of normal mucosa,adenoma andadenocarcinoma tissues from the same patient.Threesubtracted cDNA libraries were constructed and thenhybridized with forward and backward subtracted probesfor differential screening.Positive clones from eachsubtracted cDNA library were selected for sequencing andBLAST analysis.Finally,virtual Northern Blot confirmedsuch differential expression.RESULTS By this way,there were about 3-4×10~2clones identified in each subtracted cDNA library,inwhich about 85% positive clones were differentiallyscreened.Sequencing and BLAST homology searchrevealed some clones containing sequences of knowngene fragments and several possibly novel genes showingfew or no sequence homologies with any knownsequences in the database.CONCLUSION All results confirmed the effectiveness andsensitivity of SSH.The differentially expressed genesduring the development of CRC can be used to shed lighton the pathogenesis of CRC and be useful genetic markersfor early diagnosis and therapy.     

日本血吸虫消减雌性成虫cDNA文库的建立及其特异表达基因的筛选

目的 筛选雌性日本血吸虫特异表达基因。方法 感染日本血吸虫6周的家兔,用静脉灌注法收集成虫,经核糖核酸固定液固定,分别提取雌、雄成虫总RNA,纯化后获得mRNA,并反转录为cDNA。用抑制性消减杂交技术(SSH)构建雌、雄成虫正向消减(雌虫消减雄虫)及反向消减(雄虫消减雌虫)cDNA文库。用斑点杂交法筛选差异表达基因,挑选目标基因片段(与正向消减探针杂交的信号明显高于与反向消减探针杂交信号的克隆)进行测序、同源性搜索及基因功能预测分析。以日本血吸虫肌动蛋白(actin)基因作内参照,用半定量PCR(semi-quantitative PCR)鉴定目标基因在雌、雄虫体内的表达。结果 得到正向消减及反向消减cDNA文库,斑点杂交筛选出50个雌虫特异性表达的克隆,经测序得到42个表达序列标签(EST),其中,有17个基因(占40.5%)与已知日本血吸虫卵壳蛋白基因高度同源;17个基因(占40.5%)与日本血吸虫未知基因高度同源、且有一小片段与卵壳蛋白基因高度同源;有8个基因(占19.0%)与日本血吸虫其他未知基因高度同源。半定量PCR结果,6个基因在雌虫体内的表达水平明显高于雄虫,分别与GenBank的血吸虫卵壳蛋白基因AY222885、AY222895、AB017097、AF519182、M32281及血吸虫其他基因AY813556高度同源。结论 构建了雌、雄成虫正向消减及反向消减cDNA文库。用SSH可筛选日本血吸虫雌性特异性表达基因。