溴氰菊酯抗性白纹伊蚊抑制消减cDNA文库的构建

目的 构建溴氰菊酯抗性白纹伊蚊抑制消减cDNA文库。 方法 分别提取白纹伊蚊溴氰菊酯抗性株（R-lab株）和敏感株（S-lab株）总RNA，纯化后获得mRNA，并反转录为双链cDNA。以R-lab株mRNA作为实验方（tester）， S-lab株mRNA作为驱动方（driver），以及R-lab株mRNA为driver方，S-lab株mRNA为tester方，进行正、反双向抑制性消减。富集的差异表达cDNA克隆到pMD18-T载体， 构建白纹伊蚊对溴氰菊酯抗性和敏感双向消减文库。 结果 分别从正向文库和反向文库中获得580和477个阳性克隆，从所构建文库随机挑选150个克隆进行PCR鉴定，阳性克隆率为93%，cDNA片段主要分布于150～750 bp。 结论 构建了抗溴氰菊酯白纹伊蚊抑制消减cDNA文库。     

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SSH技术研究H_2O_2胁迫下细粒棘球蚴基因的表达

目的构建H2O2胁迫下细粒棘球蚴(Echinococcus granulosus)与正常组织差异表达的消减cDNA文库。方法以H2O2胁迫细粒棘球蚴cDNA为试验方(tester),正常生长的细粒棘球蚴cDNA为驱动方(driver),应用抑制性消减杂交技术(suppression subtractive hybridization,SSH)研究H2O2胁迫下细粒棘球蚴基因的表达。结果文库扩增后得到124个阳性克隆,菌落PCR分析,均得到200~1000bp插入片段。将整个文库克隆进行测序,测得序列结果利用BLAST在线软件与GenBank数据库进行同源序列比对分析和BlastX分析。结果获得重要基因的cDNA序列,如氧化还原酶、蛋白激酶、生长因子等。另有部分克隆在GenBank中无法查到对应的同源基因,可能代表了新基因。结论成功构建了H2O2胁迫与正常组织差异表达的消减cDNA文库,为研究细粒棘球蚴在抗氧化过程中的相关靶基因筛选奠定基础。     

马尔尼菲青霉酵母相消减cDNA文库的构建及初步筛选

目的构建马尔尼菲青零酵母相抑制性消减cDNA文库，寻找其在酵母相中的差异表达基因。方法分别提取马尔尼菲青零菌丝相和酵母相的总RNA并合成cDNA，然后应用抑制性消减杂交技术（SSH）。以酵母相为tester（检测子），菌丝相为driver（驱赶子），连接不同的接头，通过两轮杂交和两次抑制性PCR后，将产物与T载体连接并转染大肠杆菌。经PCR鉴定，共得到480条插入片段。序列分析和同源性比较表明一些基因与细胞壁抗原、转运蛋白、氧化还原酶等具有同源性。结果成功构建了一个以酵母相为tester（检测子）的抑制性消减cDNA文库。结论所构建的cDNA消减文库为进一步筛选马尔尼菲膏零致病相关基因奠定了基础。     

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。     

应用抑制性消减杂交技术克隆NS5A-TP4反式激活基因

目的：应用抑制性消减杂交(SSH)技术构建人类新基因NS5A-TP4反式激活基因差异表达的cDNA消减文库，克隆NS5A-TP4反式激活相关基因，了解该基因的可能生物学功能。方法：以NS5A-TP4表达质粒pcDNA3．1(-)-TP4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，从总RNA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多聚酶链反应(PCR)，将产物与pGEM-Teasy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：成功构建人类新基因NS5A-TP4反式激活基因差异表达的cDNA消减文库。文库扩增后得到63个白色克隆，进行菌落PCR分析，均得到200～1000bp插入片段。挑取含有插入片段的36个克隆进行测序，并通过生物信息学分析获得20种已知功能基因序列和5个未知功能基因。结论：应用SSH技术成功构建了NS5A-TP4反式激活基因差异表达的cDNA消减文库。该文库的建立为阐明NS5A-TP4生物学功能提供理论依据。     

抑制性消减杂交技术筛选NS5ATP13 蛋白的上调基因

目的 应用抑制性消减杂交(SSH)技术构建丙型肝炎病毒(HCV)NS5ATP13蛋白反式激活相关基因差异表达的cDNA消减文库，克隆HCV NS5ATP13蛋白反式激活靶基因。方法 以HCV NS5ATP13表达质粒pcDNA3．1(-)-NS5ATPl3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并合成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后进行测序及同源性分析。结果 成功构建人HCV-NS5ATPl3蛋白反式激活相关基因差异表达的cDNA消减文库。文库扩增后得到102个白色克隆，进行菌落PCR分析，其中96个均得到100～1000bp插入片段。挑取40个插入片段测序分析，其中2个cDNA片段为未知序列，通过生物信息学分析获得其全长序列，已被GenBank收录，另有34个为已知功能序列。结论成功筛选出2个新的cDNA序列，并获得其全长序列，可能为HCV NS5ATPl3蛋白反式激活相关靶基因。     

应用抑制性消减杂交技术筛选NS5ATP5的上调基因

目的 通过抑制性消减杂交(SSH)技术构建丙型肝炎病毒(HCV)NS5A反式激活蛋白5(NS5ATP5)的反式激活相关基因差异表达的cDNA消减文库，筛选HCV NS5ATP5蛋白反式激活靶基因。方法 以HCV NSSATP5表达质粒pcDNA3．1(-)-NSSATP5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并合成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后进行测序及同源性分析。结果 成功构建人HCV NSSATP5蛋白反式激活相关基因差异表达的cDNA消减文库。文库扩增后得到91个白色克隆，进行菌落PCR分析，其中86个均得到100～1000bp插入片段。挑取32个插入片段测序分析。其中包括结缔组织生长因子、纤维连接蛋白、胰岛素样生长因子结合蛋白1等重要的基因。结论 成功筛选出HCV NS5ATP5的上调基因。     

伯氏疟原虫青蒿素抗性产生与多药抗性基因pbmdr-1的关系

目的　研究伯氏疟原虫青蒿素抗性产生与伯氏疟原虫多药抗性基因 pbmdr- 1的关系。　方法　采集本实验室培育的抗青蒿素伯氏疟原虫阳性小鼠 (抗性指数分别为 18.9和 2 7.4 5 )血 ,制成滤纸干血滴 ,Chelex- 10 0抽提 DNA,设计引物 ,经 PCR扩增 ,连接 ,抽提质粒并用双脱氧链末端终止法测定序列 ,然后同 Gen Bank中 pbmdr- 1序列进行比较。　结果　青蒿素抗性株与敏感株均有扩增 ,且抗性株与敏感株两序列相同 ,与基因库中多药抗性基因 pbmdr- 1序列有 99%的同源性。　结论　伯氏疟原虫抗青蒿素虫株抗性产生与多药抗性基因 pbmdr- 1无明显关系     

日本血吸虫病肝纤维化小鼠肝星状细胞差异表达cDNA文库的构建和筛选

目的构建日本血吸虫病肝纤维化小鼠肝星状细胞差异表达基因的消减cDNA文库,并筛选差异表达基因。方法取日本血吸虫尾蚴经腹部感染小鼠致肝纤维化。采用抑制性消减杂交技术(SSH),分离小鼠肝星状细胞(HSC)及正常小鼠HSC,通过对比寻找差异表达基因。将其与T载体连接(T/A克隆),其产物转化大肠埃希菌DH5α,经文库扩增后,随机挑取白色克隆进行酶切鉴定,克隆经过正、反向杂交,阳性克隆经过测序和BLAST(局部相似性基本查询工具)进行表达序列标签(ESTs)差异基因分析。最后经模拟Northern印迹确认基因表达差异。结果扩增消减cDNA文库获得400余个白色阳性克隆,随机挑取的克隆经酶切鉴定后均有200～600 bp插入片段。ESTs分析获得76个序列,其中70个序列提示与血吸虫病肝纤维化或与其相关的基因,6个在公共数据库中未找到同源序列片段。结论用SSH法及T/A克隆技术成功构建了肝纤维化小鼠HSC与正常小鼠HSC差异表达基因的消减cDNA文库。     

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.     

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。     

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.     

丙型肝炎病毒非结构蛋白NS4B反式激活基因的克隆化研究

目的应用抑制性消减杂交(suppression subtractive hybridization,SSH)技术构建丙型肝炎病毒(HCV)非结构蛋白4B(NS4B)转染细胞差异表达cDNA消减文库,克隆HCV NS4B蛋白反式激活相关基因.方法以HCV NS4B表达质粒pcDNA3.1(-)-NS4B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,提取mRNA并逆转录为cDNA,进行抑制性消减杂交分析.将富集的二次PCR产物与T/A载体连接,并转染大肠杆菌进行文库扩增,随机挑取克隆聚合酶链反应(PCR)扩增后进行测序及同源性分析.结果文库扩增后得到33个阳性克隆,经菌落PCR分析显示其中28个克隆含有大小不等的200～1000 bp插入片段.测序及同源性分析显示,12种已知基因编码蛋白,包括一些与细胞周期、信号传导及肿瘤发生等细胞生长调节密切相关的蛋白编码基因,可能是NS4B反式激活靶基因.结论成功构建了HCV NS4B反式激活基因差异表达的cDNA消减文库,为进一步阐明HCV NS4B反式调节的靶基因在肝炎、肝纤维化和肝细胞癌发生的分子生物学机制提供理论依据.     

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.     

Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma

AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database. METHODS: Human VECs related to gastric adenocarcinoma and corresponding normal tissue were separated by magnetic beads coupled with antibody CD31 (Dynabeads CD31). A few amount of total RNA were synthesized and amplified by SMART PCR cDNA Synthesis Kit. Then, using SSH and T/A cloning techniques, cDNA fragments of differentially expressed genes in human VECs of gastric adenocarcinoma were inserted into JM109 bacteria. One hundred positive bacteria clones were randomly picked and identified by colony PCR method. To analyze cDNA libraries of gastrocarcinoma and VECs in CGAP database, the tools of Library Finder, cDNA xProfiler, Digital GENE Expression Displayer (DGED), and Digital Differential Display (DDD) were used. RESULTS: Forward and reverse subtraction cDNA libraries of human VECs related to gastrocarcinoma were constructed successfully with SSH and T/A cloning techniques. Analysis of CGAP database indicated that no appropriate library of VECs related to carcinoma was constructed. CONCLUSION: Construction of subtraction cDNA libraries of human VECs related to gastrocarcinoma was successful and necessary, which laid a foundation for screening and cloning new and specific genes of VECs related to gastrocarcinoma.