高、低淋巴道转移能力小鼠肝癌细胞株抑制性消减杂交文库的构建

应用抑制性消减杂交（suppression subtracted hybridization,SSH）技术,分别构建2个不同淋巴道转移能力小鼠肝癌细胞株的SSH文库,高转移文库以高转移能力细胞株Hca-F为检测子（tester）,同源的低转移能力细胞株Hca-P为驱赶子（driver）;低转移文库则相反.随机筛选2个文库阳性克隆进行测序,并在GenBank数据库中进行同源性比较.成功构建高、低淋巴道转移能力小鼠肝癌细胞株SSH文库,高、低转移文库中分别包含995个及967个阳性克隆;其中,95%的阳性克隆含有300bp～1 000bp不等的插入片段;随机测序显示,文库中含有已知的基因、ESTs及与任何序列无同源性的新基因片段.     

食管癌相关基因1编码蛋白的相互作用蛋白筛选

目的　筛选与食管癌相关基因 1(ECRG 1)编码蛋白相互作用的蛋白 ,为其功能研究奠定基础。方法　将编码ECRG 1羧基端 378个氨基酸的DNA序列插入到pGBKT7 DNA BD载体 ,与编码Gal4DNA结合结构域的DNA序列拼接做融合基因 ,将此重组质粒与已克隆到pACT2载体上的人肝脏cDNA文库 (与编码Gal4激活结构域的DNA序列融合 )共转化酵母细胞AH10 9。ECRG 1与相应的人肝脏cDNA片段编码的蛋白发生相互作用后 ,可激活报告基因的表达。排除假阳性后 ,阳性克隆中的文库cDNA进行测序分析。同源性检索搜寻GenBank中与之相同或相似的序列。结果　共转化得到约 3× 10 6个转化子 ,2 3个克隆有报告基因的表达。排除假阳性后 ,得到 2个阳性克隆 ,它们分别编码Miz 1(myc interactingznfingerprotein 1)和FLNA(actin bindingprotein 2 80 )。结论　ECRG 1基因编码蛋白在酵母中可特异性的结合Miz 1和FLNA ,提示ECRG 1可能通过与MIZ 1、FLNA相互作用参与调控细胞周期的运行。     

人胃印戒细胞癌组织差异表达基因克隆的研究

目的：分离胃印戒细胞癌组织差异表达基因。探讨人胃印戒细胞癌发生的分子机制。方法：采用抑制性消减杂交技术(SSH)．构建人胃印戒细胞癌组织cDNA消减文库；随机挑选部分阳性克隆测序，与GenBank中的已知序列进行基因同源性分析，并进一步探讨基因功能。结果：成功构建高消减效率的人胃印戒细胞癌组织cDNA消减文库，并分离出多个差异表达基因片段：其中1条与染色体序列相似，可能代表未知的新基因序列，已被GenBank接收(注册号：CN446913)；其他多为已知的肿瘤相关基因的部分片段。结论：1)SSH技术是分离差异表达基因的有效方法；2)胃印戒细胞癌的发生发展与多种基因表达异常有关。     

人胃印戒细胞癌组织差异表达基因克隆的研究

目的:分离胃印戒细胞癌组织差异表达基因,探讨人胃印戒细胞癌发生的分子机制。方法:采用抑制性消减杂交技术(SSH),构建人胃印戒细胞癌组织cDNA 消减文库;随机挑选部分阳性克隆测序,与GenBank中的已知序列进行基因同源性分析,并进一步探讨基因功能。结果:成功构建高消减效率的人胃印戒细胞癌组织cDNA消减文库,并分离出多个差异表达基因片段:其中1 条与染色体序列相似,可能代表未知的新基因序列,已被GenBank 接收(注册号:CN446913);其他多为已知的肿瘤相关基因的部分片段。结论: 1 )SSH技术是分离差异表达基因的有效方法;2)胃印戒细胞癌的发生发展与多种基因表达异常有关;     

高淋巴系统转移能力小鼠肝癌细胞株差异表达基因的筛选

目的 筛选高淋巴系统转移能力小鼠肝癌细胞株差异性表达基因。方法 应用抑制性消减杂交(SSH)技术,构建高淋巴系统转移能力小鼠肝癌细胞株Hca-F及其同源低转移细胞系Hca-P的cDNA消减杂交文库,筛选阳性克隆进行测序,并在GenBank数据库中进行同源性比较。结果 成功克隆10个差异基因片段,其中2个为新基因。结论 SSH技术是克隆差异表达基因及发现新基因的有效方法。     

人前列腺癌高低转移细胞株差异表达基因的筛选

目的：筛选高低转移能力人前列腺癌细胞株PC3M-1E8和PC3M-2B4间差异表达基因。方法：应用抑制性消减杂交（SSH）技术,对高转移能力人前列腺癌细胞株PC3M-1E8及其同源低转移细胞株PC3M-2B4进行2次消减杂交,第1次SSH实验,以PC3M-2B4细胞株为实验方,PC3M-1E8株为驱动方,构建正向消减文库。第2次实验则以PC3M-1E8株为实验方,PC3M-2B4为驱动方,构建反向消减文库。筛选出来的阳性克隆进行测序,并在GeneBank数据库中进行同源性比较后从中选取若干序列进行实时定量PCR验证。结果：2个消减文库共得到238个阳性克隆,从中各随机选取8个克隆测序并进行同源性分析,发现其中12条序列来自于11个已知基因,另外4条序列为新的未知功能的基因序列标签（EST）。结论：成功构建了高低转移力人前列腺癌细胞株的双向消减杂交文库。     

肺癌转移相关基因的差减文库的构建

目的发现与肺癌转移相关的基因.方法选择4例转移和5例非转移患者肺癌的RNA混合样品进行差减杂交.经过两步骤的杂交后,构建了转移相关基因的差减文库.对获得阳性克隆进行测序和同源性分析.结果一共获得了225个阳性克隆.在GenBank中序列比对的结果表明,它们分属124个基因.其中25个基因出现的频率大于一次,并有9个序列未找到同源基因.结论该结果将有助于各类肿瘤转移相关基因的研究.     

肿瘤特异性抗原超家族新成员apr-1基因的克隆及亚细胞定位

背景与目的apr1是我们在凋亡的人早幼粒白血病细胞HL-60中克隆的一个人类新基因,初步研究表明它可能是一个凋亡相关基因(GenBank ID NM_014061)。为进一步了解该基因的功能背景,我们克隆了该基因并进行了生物信息学分析和亚细胞定位。方法利用逆转录PCR方法克隆该基因的cDNA序列,测序后利用生物信息学方法进行了基因组定位、氨基酸保守结构域搜索、同源蛋白比较和基因进化树分析;并进行了该基因的亚细胞定位。结果apr.1定位于人染色体上的Xp11.22,有两个MAGE家族结构域,基因进化上同源于MAGE.D1和Necdin,基因表达产物定位于真核细胞的细胞核。结论apr.1属于MAGE家族的一个新分子,可能属于Ⅱ类MAGE基因。     

NAG14基因结构域蛋白的原核表达

基因组测序完成在即 , 运用已有的基因数据库 , 人们已经克隆了大量与人类重大疾病和肿瘤 相关的基因 . 结构基因组研究的深入 , 要求进一步分析新克隆基因的功能以及其结构与功能 之间的关系 . 我们应用定位-候选克隆策略结合生物信息学手段筛选与鼻咽癌负相关的 EST(Expressed Sequence Tags), cDNA克隆测序和 5′ RACE扩增获取候选 EST的 cDNA序列 , 已 克隆到一个新基因 NAG14 (GenBank登录号 : AF196976), 该基因在鼻咽癌细胞株 HNE1和 75% 活 检组织表达缺失或下调 [1]. 结构域是蛋白质在一级结构和三级结构之间的结构单位 , 也是生 物大分子生物功能的结构单位 [2]. 通过对蛋白质结构域的分析 , 并分离结构域 , 对分离的结 构域的结构和功能进行研究 , 可以对蛋白质功能有一个更为深入的结构层次的了解 . 为了解 NAG14基因所编码蛋白质的功能 , 我们用多聚酶链式反应 (PCR)方法分离出 NAG14基因的亮氨酸 重复区 ( leucine repeat pegion, LRR) 结构和 IgC2 结构域蛋白编码区序列 , 构建 LRR结构 域和 IgC2结构域的原核表达载体 , 在大肠杆菌中诱导这两个结构域蛋白的表达 , 为进一步制 备抗 NAG14基因蛋白的多克隆抗体和 NAG14基因功能研究打下基础 .     

应用抑制性消减杂交技术克隆人肺鳞癌差异性表达基因

目的 克隆人肺鳞癌肿瘤差异性表达基因。方法 应用抑制性消减杂交技术（suppression subtractive hybridization,SSH)构建人肺鳞癌cDNA消减杂交文库，筛选后挑选阳性克隆进行测序，并在Gen Bank数据库中进行同源性比较，最后经RT－PCR验证。结果 成功克隆10个差异基因片段，其中2个为新基因（GenBank登录号分别为：C20：AF363068；C34：AY032661）。结论 抑制性消减杂交技术是克隆差异表达基因及发现新基因的有效方法。     

一种新的黑色素瘤相关抗原MAGE-C2的基因克隆及表达

目的:克隆人黑色素瘤相关抗原MAGE-C2的cDNA,构建真核、原核表达载体并转入相应细胞获得表达.方法:采用RT-PCR技术,从直肠癌细胞系SW480的总RNA中克隆了MAGE-C2 cDNA序列,并将开放读框分别插入质粒pEGFP-C1和pGEX-4T-3中,获得pEGFP-MAGE-C2绿色荧光蛋白(GFP)融合表达载体和pGEX-MAGE-C2谷胱甘肽转移酶(GST)融合蛋白表达载体,将pEGFP-MAGE-C2转染293T细胞,观察绿色荧光融合蛋白在细胞中的定位;将pGEX-MAGE-C2转化大肠杆菌BL21株,经IPTG诱导表达GST融合蛋白,并用谷胱甘肽亲和层析法纯化重组蛋白.结果:获得MAGE-C2开放读框并构建表达载体,测序结果与GenBank收录的序列相一致.真核表达载体转染293T细胞后显示MAGE-C2蛋白定位于细胞核;原核重组蛋白大部分以可溶性形式表达,亲和层析后,获得了较高纯度的MAGE-C2-GST融合蛋白.分析显示原核表达GST融合蛋白的相对分子质量为70 000,使用抗GST单克隆抗体进行Western blot分析证实为目的蛋白.结论:成功的获得MAGE-C2基因,构建了其表达载体并获得表达,为进一步制备MAGE-C2特异性单抗及深入探讨MAGE-C2可能参与肿瘤发生发展的机制提供了重要条件.     

Complete nucleotide sequence of mouse mammary tumor virus from jyg chinese wild mice: Absence of bacterial insertion sequences in the cloned viral gag gene

Mammary tumors of a newly isolated strain of Chinese wild mouse (JYG mouse) harbor exogenous mouse mammary tumor virus (MMTV). The complete nucleotide sequence of exogenous JYG-MMTV was determined on the proviral 5' long terminal repeat (LTR)(partial)-gag-pol-env-3' LTR (partial) fragment cloned into a plasmid vector and the cDNA sequence from JYG-MMTV producing cells. Similarly to the other MMTV species the LTR of JYG-MMTV contains an open reading frame (ORF). The amino acid sequence of the JYG-MMTV ORF resembles that of SW-MMTV (92% identity) and endogenous Mtv-7 (93% identity) especially at the C-terminal region. Thus, a functional similarity in T-cell receptor V beta recognition as a superantigen is implicated among these MMTV species. Analysis of the viral gag nucleotide sequence revealed that this gene is not disrupted by the bacterial insertion sequence IS1 or IS2, which have been reported to be present in the majority of the plasmids containing the gag region. Comparison of amino acid sequences of JYG-MMTV with those of BR6-MMTV showed that over 96% of the amino acids of gag, pol, protease and env products are identical. These results suggest the intact nature of the nucleotide sequence of the near full-length MMTV genome cloned in the plasmid.     

肺癌相关基因HLCDG1的克隆和表达分析

背景与目的：比较基因组杂交和微卫星多态性位点全基因组扫描结果显示肺癌患者在染色体3p、5q、6q、9p、10q、llp、13q、17p、19p等区域存在高频率的杂合性缺失,提示可能有多个未知的易感基因或抑癌基因与肺癌的发生、发展相关。本研究选用在mRNA差异显示研究中获得的在肺癌组织中表达下调且代表新基因的表达序列标签(expressed sequence tag,EST)LXDDl为进一步研究对象,克隆其全长cDNA。方法：采用Northern杂交验证LXDDl在肺癌中的表达差异。并用MTN(Multiple Tissue Northern Blots^TM)膜检测其在正常组织中的表达及其所代表基因的转录本大小;克隆其全长cDNA,并利用生物信息学对该序列进行初步分析;用差异RT-PCR检测新基因在肺癌组织、配对癌旁非癌组织、肺癌细胞系和其它肿瘤细胞系中的表达。结果：成功地克隆了一个在肺癌中表达下调的新基因。HLCDGl(human lung carcinoma deleted gene 1),GenBank登录号为AF447582,全长cDNA为3．113kb,预测其开放阅读框编码一个含166个氨基酸的跨膜蛋白质。通过电子-聚合酶链反应(electric-polymerase chain reaction,e-PCR)将该基因定位于5q33。结论：HLCDGl基因是一个在肺癌中表达下调的新基因。这提示HLCDGl可能与肺癌的发生、发展相关。     

The cloning of hrnt-1 using a combination of cdna library screening with biotin-labeled probe and rapid amplification of cDNA ends

Although the physical map of human genome will come to an end and has assisted directly in identifying about 100 disease-causing genes, as well as providing an excellent framework for the complete sequencing of the human genome, one of the most difficult challenges ahead is to find and study genes involved in diseases that have a complex pattern of inheritance, such as those that contribute to diabetes, asthma, cancer and mental illness. It is still necessary to develop an effective method to c…     

Lewis肺癌的RAPD分析及其差异片段克隆

目的 检测Lewis肺癌的遗传改变,寻找与癌变相关的DNA序列或片段。方法 用105条引物对Lewis肺癌及其来源小鼠C57BL／6J正常组织基因组DNA进行随机扩增多态DNA（RAPD）扩增,比较肿瘤组织与正常组织的扩增图,对差异很明显的片段回收、克隆和测序,序列与GenBank数据库进行同源性比较。结果 105条引物中有25条引物扩增出的条带在肿瘤组织与其相应正常组织间存在差异。引物AB7－2和AB7－11扩增所得差异片段L7－2和L7－11回收纯化后克隆测序。L7－2片段长730bp,该序列与已知序列没有任何相关性。L7－11长779bp,与小鼠的V kappa 21-11gene在核酸水平具有90％的同源性。结论 本研究用RAPD技术检测出Lewis肺癌存在一定程度的遗传改变,并发现与Lewis肺癌发生相关的DNA序列L7－2和L7－11,这种改变可能与肺癌的发生相关。     

Extranodal marginal zone B-cell lymphoma genotyping by Alu-polymerase chain reaction

DNA amplification by polymerase chain reaction (PCR) with primers designed on the widely distributed Alu sequences allows the production of specific inter-Alu DNA-fingerprints. Amplification of tumour and matched normal DNA can show differences due to genetic alterations within the tumour genome. We applied this approach to study low-grade extranodal marginal zone B-cell lymphoma (of MALT type). After digestion with restriction enzymes, DNA samples were separately amplified by PCR with three different Alu-primers. A comparison between the fingerprint pattern from lymphoma and normal samples was made. Inter-Alu bands differing between the two samples were excised from the gel, cloned and sequenced. Nine cases of low-grade MALT-lymphomas have been analysed, giving seventeen different bands between tumour and normal. DNA sequence analysis showed identities for three of them with sequences available at the GenBank. The methodology of Alu-PCR to detect DNA-based abnormalities, in addition or combination with RNA-based methods, is a powerful tool to identify candidate regions frequently altered in tumours. With the increased available genomic sequences through the Human Genome Project, there will be an increasing probability of picking up perfect homologies with these sequences using cloned differential Alu-PCR bands in BLAST searches through genome databases.     

Assignment of the gene for human DNA polymerase beta (POLB) to chromosome 8

A cloned cDNA segment of 442 bp which contains the region coding the N-terminal sequence of rat DNA polymerase beta polypeptide was used as a probe in Southern hybridization analysis to identify the gene for this enzyme. This probe hybridized with DNA from mouse and human in addition to rat DNA, and the BamHI fragment containing DNA polymerase beta gene sequence was unique in size in each species: 15.8 kb for rat DNA, 8.7 kb for mouse DNA and 10.1 kb for human DNA. DNA extracted from a panel of 12 independent human-mouse somatic cell hybrids was analyzed by Southern blot hybridization with the cloned cDNA probe. The results indicate that the gene for the human DNA polymerase beta sequence (POLB) is located on chromosome 8.     

Isolation and partial characterization of a transformation-associated sequence from human nasopharyngeal carcinoma.

A transforming activity associated with Chinese nasopharyngeal carcinoma (NPC) cell line CNE2 DNA has been identified by transfer into nontransformed promotion-sensitive mouse JB6(P+) C141 cells. To clone this transformation-associated sequence, we carried out three cycles of transfection, followed by cloning of anchorage-independent transformants in soft agar. A tertiary CNE/JB6 clonal transfectant cell line 625 whose DNA showed transforming activity, as indicated in both soft-agar assay and nude-mice implantation, was used to make a genomic library in the vector lambda dash. Using the human repeated sequence Blur 8 to screen the library, we obtained 10 human Alu-positive clones. A cloned Alu-positive insert of 16 kbp, CNE 323, was characterized in detail. CNE 323 transferred moderate transforming activity when introduced into JB6 P+ cells and showed no homology to Ha-, Ki-, or N-ras genes; human promotion sensitivity genes; src, myb, jun, myc, fos, raf, or int-2 oncogenes; or epidermal growth factor receptor. The isolated CNE 323 DNA sequence appeared to preserve the genomic structure of the original sequence found in CNE2 cells and in nude mouse tumors induced by CNE2 cells or by CNE/JB6 transfectant cells, indicating that the cloned NPC sequence was activated during NPC carcinogenesis and not during transfection or construction of the library, and that the cloned sequence or a larger sequence of which it was part played a role in tumor formation. Finally, we identified a 1.3-kb mRNA that hybridizes to a subclone of the 16-kb NPC sequence in CNE2 cell poly (A)+ RNA.