锌指蛋白ZNF580定位在MGC803细胞核

新基因(AF184939)ZNF580是由本组克隆并于Genbank注册的C2H2型锌指蛋白转录因子基因,其cDNA 748~1266 bp之间的碱基构成一个完整的开放阅读框,编码172个氨基酸。蛋白功能基序分析表明:其羧基端序列中含有3个重复串联的C2H2型锌指蛋白主构域:CX2CX3FX5LX2HX3H(X代表保守性差的氨基酸),富含脯氨酸残基的氨基端序列为转录激活结构域[1]。利用绿色荧光(green fluorescence pro-tein,GFP)在活细胞内的示踪作用[2],我们将GFP与ZNF580cDNA开读框融合,导入胃癌MGC803细胞株中,直观地观察到ZNF580基因在细胞内的表达及亚细胞定位,…     

ZNF580-EGFP融合蛋白的亚细胞定位研究

目的：构建增强型绿色荧光报告蛋白(EGFP)与人ZNF580融合蛋白的真核表达载体，转染MGC803细胞进行表达，研究ZNF580-EGFP融合蛋白在MGC803细胞的亚细胞定位。方法：利用PCR技术扩增ZNF580基因cDNA开放阅读框架全编码区、N端编码区、C端编码区，分别克隆到真核表达载体pEGFP-C1，BglⅡ及HindⅢ双酶切电泳筛选、鉴定并测序。荧光显微镜下观察ZNF580-EGFP在MGC803细胞中的表达及亚细胞定位。结果：酶切pEGFP-ZNF580(1-172)、pEGFP-ZNF580(1-93)、pEGFP- ZNF580(94-172)，电泳分析插入片段长度分别为：526 bp、289 bp、247 bp，经连接点两端进行测序证实连接正确。pEGFP-ZNF580(1-172)、pEGFP-ZNF580(94-172)表达的ZNF580-EGFP融合蛋白定位在MGC803细胞核。结论：成功构建真核表达载体并在MGC803细胞中得到表达，分析其核定位信号可能位于ZNF580蛋白的C端C2H2型锌指主构域94-172位氨基酸区间。     

锌指基因217与肿瘤

锌指基因217(zinc finger gene217,ZNF217)定位于染色体20q13.2,在各种肿瘤如乳腺癌、卵巢癌、胃癌中过度扩增,且与肿瘤的浸润性关系密切。ZNF217的过度扩增能减弱由端粒功能障碍和阿霉素引起的凋亡信号,引起Akt的磷酸化增加,促进肿瘤的发生和生长。抑制ZNF217能增加细胞对阿霉素的敏感性。ZNF217是一转录阻遏蛋白,能募集CtBP1/2并通过其锌指结构与启动子相结合,调节基因的转录。这些基因与细胞分化和增殖有关。目前由于缺乏明确的ZNF217靶向基因,ZNF217的功能尚不完全清楚。     

生物化学和分子生物学

0500591 包含BTB锌指结构的新基因Bsg2结构特性及其在发育阶段的组织表达特异性，0500592 HeLa细胞中Skp2的表达调控p21^WAF/CIP1稳定性,0500593 登革病毒衣壳蛋白靶向核酸酶表达系统的建立及应用,0500594 转录因子XBP-1的融合表达、纯化及多克隆抗体的制备,0500595 N端缺失突变对核糖核酸酶抑制因子活性的影响.     

肝癌患者血清和癌组织中锌指蛋白216表达增加

目的 寻找肝癌相关抗原.方法 应用SEREX技术对肝癌组织cDNA文库进行血清学筛选,获得人类锌指蛋白ZNF216.利用原核表达和亲和层析技术获得ZNF216的6*His融合蛋白,建立间接ELISA方法,检测血清中产生的ZNF216抗体水平;利用RT-PCR技术检测肝癌患者肝癌组织及癌旁组织内ZNF216 mRNA的水平.结果 肝癌患者血清中产生的抗ZNF216抗体水平高于正常个体,ZNF216 mRNA在肝癌组织内的表达明显高于癌旁组织.结论 ZNF216在肝癌病人血清及癌组织内的过表达可能参与肝癌的发生,但还需要进一步研究证明.     

ADAR1通过RNA编辑上调ZNF655表达并促进人肝癌细胞系HepG2中HBV复制

目的探讨细胞中ADAR1对锌指蛋白ZNF655的作用及其对乙肝病毒(HBV)复制的影响。方法在人肝癌细胞系HepG2细胞中,利用桑格测序验证ZNF655 3'UTR存在ADAR1 RNA编辑位点;RT-qPCR检测ADAR1和ZNF655 mRNA的表达及HBV total RNA和HBV 3.5 kb RNA的表达;双荧光素酶报告基因检测荧光素酶的相对表达;Western blot检测ADAR1和ZNF655蛋白的表达;ELISA检测ZNF655对HBV标志物HBs Ag和HBe Ag的影响。结果 ZNF655基因3'UTR上chr7:99575277位点在DNA水平为纯合型,在RNA水平为杂合型;ZNF655基因3'UTR上chr7:99575277位点的编辑型G比正常型A的荧光素酶活性显著升高(P<0.001);在转录和翻译水平,ADAR1都显著增加了ZNF655的表达(P<0.001);ZNF655对HBV的表达起到促进的作用。结论 ADAR1通过编辑ZNF655的3'UTR上的chr7:99575277位点,使编辑位点A转换成G,上调了基因的表达,进而起到对HBV复制的促进作用。     

应用荧光原位杂交技术检测卵巢浆液性囊腺癌中ZNF217基因拷贝数

目的探讨锌指蛋白217（zinc finger protein 217，ZNF217）基因在卵巢浆液性囊腺癌中20号染色体基因拷贝数改变情况及其临床意义。方法应用荧光原位杂交技术及LSI ZNF217探针对2种卵巢癌细胞株SKOV3、HO-8910和23例卵巢浆液性囊腺癌、10例浆液性囊腺瘤及7份正常卵巢组织进行检测。结果两种卵巢癌细胞株及12例卵巢癌出现ZNF217基因扩增，浆液性囊腺瘤有1例发生了基因的扩增，其余的及正常卵巢组织未出现基因扩增，卵巢癌与卵巢浆液性囊腺瘤、正常卵巢细胞相比较，ZNF217基因拷贝数改变差异有统计学意义（P〈0．05），而低分化的卵巢癌比高分化发生ZNF217基因扩增的几率明显增高（P〈0．05），Ⅲ～Ⅳ期比Ⅰ期的卵巢癌ZNF217基因拷贝数明显增多（P〈0．05）。结论ZNF217基因很可能是卵巢癌发生的促进因子之一，并与卵巢癌分化及预后不良有关。     

ZNF521促进结肠癌的进展且受miR-211-5p靶向调控

目的:探索锌指蛋白521(ZNF521)在结肠癌中的表达、功能和临床意义,并研究其机制.方法:采用免疫组织化学、蛋白质印迹法和qRT-PCR检测结肠癌组织或细胞系中ZNF521或miR-211-5p的表达;采用小干扰RNA(small interfering,siRNA)和miRNA模拟物分别构建ZNF521低表达和m...     

HLA—Ⅱ类抗原的表达调控及其在肿瘤组织中的表达

HLA－Ⅱ类抗原在免疫应答和免疫调节中具有重要作用，其表达调节主要发生在转录水平上。在它的启动子区内存在W盒、Y盒、X盒等顺式作用元件，调节HLA－Ⅱ类抗原的表达。HLA－Ⅱ类抗原启动子区具有的高度多态性，也可通过多种途径影响HLA－Ⅱ基因的表达。肿瘤细胞可异常表达HLA－Ⅱ类抗原，干扰素、启动子多态性、CⅡTA、RB蛋白等多种因素影响其表达。     

一种筛选原核高效表达克隆的方法

外源基因在大肠杆菌中的低表达往往与基因５’端核苷酸序列不利于翻译起始有关。未经修饰的ＧＭ－ＣＳＦ在大肠杆菌中表达量很低，我们选用ＧＭ－ＣＳＦ作为报道基因，在不改变氨基酸序列的前提下，通过聚合酶链反应（ＰＣＲ）扩增，构建了两类包含５’端１０个密码子的简并序列库，为便于快速筛选高表达的克隆，构建了四环素抗性（ＴｅｔＲ）选择载体筛选系统，其基本原理是外源基因与ＴｅｔＲ基因串联，高表达的ＧＭ－ＣＳＦ５’端带动ＴｅｔＲ的表达，逐渐提高四环素浓度就可以筛选出适合高表达的ＧＭ－ＣＳＦ５’端序列。最终在四环素浓度高达６０μｇ／ｍｌ的培养基上筛选了数个高表达克隆。这种系统对大肠杆菌中表达量低、但具有重大经济价值的细胞因子的表达有重要的意义     

Localization of a novel zinc finger gene to the human chromosome 7p11.2-p12 by fluorescencein situ hybridization

A novel zinc finger gene (ZNF182) was isolated from a human fetal cardiac cDNA library, using a consensus C2H2 zinc finger oligonucleotide probe. This gene was assigned to human chromosome 7p11.1-p12 by fluorescencein situ hybridization (FISH). Additional FISH signals were identified on both the long and the short arms of chromosome 19, suggesting the presence of homologous genes at these loci.     

Identification of ZNF200 as a novel binding partner of histone H3 methyltransferase G9a

G9a belongs to the subfamily of histone H3 lysine 9 (H3-K9)-specific methyltransferases. On amino acid sequence alignment of human and Drosophila G9a, we found that the N-terminal region from amino acids 532-605 to be evolutionarily conserved and named this the G9a homology domain (GHD). Using the GHD of human G9a (hG9a) as a bait, we isolated cDNA encoding a zinc finger protein 200 (ZNF200), which contains five C(2)H(2)-type zinc finger domains in tandem arrays. Interaction between G9a and ZNF200 could be demonstrated by in vitro binding assays and immunoprecipitation experiments using cultured human HEK293 cell extracts. GST pull-down assays using deletion derivatives of ZNF200 revealed that the interaction is through a region encompassing three of the five zinc finger domains. Furthermore, ZNF200 appear to co-localize with G9a in the nucleoplasm of HEK293 cells as discrete speckles. These results demonstrate that ZNF200 is a novel binding partner of G9a.     

ZNF268, a novel kruppel-like zinc finger protein, is implicated in early human liver development

The advancement in gene knockout and transgenesis have brought about enormous improvement in our understanding of mouse embryogenesis in the past decade or so. On the other hand, relatively little is known about human embryogenesis due largely to the lack of easy access to human embryos and tissues for biomedical studies. We have previously isolated a novel zinc finger gene, ZNF268, from a 3-week-old human embryo cDNA library in an effort to identify genes important for human embryonic development. To investigate the potential involvement of ZNF268 in human embryogenesis, we report here the spatial and temporal regulation of its expression during development. Northern blot and Western blot analyses revealed that ZNF268 is expressed in early embryos, predominantly, if not exclusively, in fetal liver with little detectable expression in other fetal organs. Interestingly, unlike most zinc finger proteins, ZNF268 protein was found to be localized mainly in the cytoplasm of embryonic hepatocytes. This subcellular localization was substantiated by the localization of EGFP-ZNF268 fusion protein overexpressed in the transfected COS7 cells. These results suggest that ZNF268 plays a role in early human liver development most likely by functioning through a cytoplasmic mechanism.     

Identification and characterization of SKAT-2, a novel Th2-specific zinc finger gene

We have identified a novel Kruppel-type zinc finger (ZF) gene, SKAT-2, which is selectively expressed by murine Th2 cells. The protein encoded by this gene has 14 C2H2-type ZF tandemly arrayed at its C terminus and N-terminal SCAN box and KRAB domains. SKAT-2 is tissue restricted in expression at the RNA level, detectable only in brain and at low levels in kidney and spleen and few hematopoietic cell lines. By in situ hybridization, SKAT-2 expression was found to peak in antigen-stimulated CD4(+) T cells after 2-3 days of culture under Th2 but not Th1 biasing conditions. This pattern of expression closely mirrored that of GATA-3 in the same cells. In transient transfection experiments in phorbol 12-myristate 13-acetate/ionomycin-stimulated EL4 cells, SKAT-2 was found to up-regulate the activity of the IL-4 but not the IL-5 promoter, contrasting with the ability of GATA-3 to activate both promoters. This result was confirmed using clones of EL4 cells stably expressing an inducible form of SKAT-2, thus SKAT-2 is a novel Th2-specific gene that may play a role in selective regulation of cytokine genes in T cells.     

ZNF580在1-磷酸鞘氨醇诱导内皮细胞迁移和增殖中的作用

 目的：研究人锌指蛋白ZNF580在1-磷酸鞘氨醇(sphingosine 1-phosphate, S1P)诱导内皮细胞迁移和增殖中的作用,为探讨ZNF580功能提供科学依据。方法：RT-PCR检测S1P受体在EA.hy926细胞的表达情况;不同浓度(0~10 μmol/L) S1P刺激EA.hy926细胞不同时间(0~12 h)后,RT-PCR和Western blotting检测S1P对ZNF580表达的影响;利用p38 MAPK信号通路特异性抑制剂SB203580研究S1P是否通过此信号通路影响ZNF580的表达;脂质体转染法获得瞬时过表达和瞬时低表达ZNF580的EA.hy926细胞;Transwell实验及MTT比色法分析ZNF580对内皮细胞迁移和增殖活性的影响。结果：EA.hy926细胞表达S1P1、S1P3和S1P5三种受体,其cDNA的特异性扩增产物分别为352 bp、701 bp和236 bp;S1P刺激EA.hy926细胞后,ZNF580的表达呈现剂量和时间依赖性增高;SB203580能够抑制S1P诱导的ZNF580的上调作用;ZNF580过表达（低表达）后内皮细胞迁移和增殖活性明显增强（减弱）。结论：S1P通过p38 MAPK信号通路影响ZNF580的表达;ZNF580在内皮细胞迁移和增殖过程中起着重要的调控作用。