寡聚蛋白Trim34α对NF-κB报告基因的负向调节作用

目的 测Trim34α对TAB2诱导的NF-κB报告基因活化的影响.结果 经鉴定成功构建Trim34α真核表达载体,该载体表达的Trim34α蛋白能相互聚集形成寡聚体(Trim小体);Trim34α可显著抑制TAB2诱导的NF-κB荧光素酶报告基因活化.结论 Trim34α可以在胞内形成寡聚体,Trim34α能显著抑制TAB2诱导的NF-κB报告基因活化.

Abstract：

Objective To investigate the effects of Trim34α on the activation of luciferase reporter gene containing NF-κB promoter induced by adaptor proteins TAB2. Methods The total RNA was isolated from HeLa cells. After amplification with RT-PCR, the target sequences were cloned into 5'-Flag-pcDNA3.1 (+) vector. The recombinant vector was confirmed by restriction enzyme digestion, colony PCR and sequencing. It was transfected into HEK293T cells to detected Trim34α expression by Western blot. Simultaneously, the effects of Trim34α on the NF-κB activation induced by TAB2 were determined by dual-luciferase reporter assay. Results Restriction enzyme digestion, colony PCR and sequencing confirmed the vector was constructed successfully, furthermore it expressed Trim34α protein in HEK293T cells. Moreover, trim34α could form high-molecular-weight oligomeric protein, and here we called it trimsome. Interestingly, dual-luciferase assay showed that Trim34α could effectively block TAB2-induced NF-κB activation. Conclusion Trim34α was involved in negative regulation of TAB2-induced NF-κB activation and could form high-molecular-weight oligomer.     

人大麻素受体启动子真核报告质粒的构建及活性分析

目的:利用双荧光素酶报告基因体系,根据大麻素受体1(cannabinoid receptor 1,cnr1)启动子序列不同部位的转录活性来初步确定其活性区域,为脊髓缺血耐受保护中cnr1高表达的转录调控的研究奠定基础。方法:从NCBI中获取cnr1基因转录起始点5’端向上约1800 bp的核苷酸序列,按照300 bp的距离间隔,设计6个不同长度的截短体PCR引物,以人全血基因组DNA为模板,分别扩增cnr1启动子区域的截短体片段,并克隆入荧光素酶报告基因pGL3-Basic质粒中。将含有不同截短体的重组质粒分别转染Hela、Jurket和A549细胞后行荧光素酶活性检测。根据不同截短体转录活性检测结果,确定cnr1启动子活性区域。结果:成功将cnr1启动子区6个不同长度(1800、1500、1200、900、600和300 bp)的截短体克隆入荧光素酶报告基因pGL3-Basic质粒。在3种细胞系Hela、Jurket和A549的荧光素酶活性检测均显示600 bp的截短体转录活性最强。结论:成功构建了cnr1启动子的报告基因重组质粒,初步证实-600 bp到-200 bp区为cnr1的启动子的活性区域,从而为进一步研究cnr1的转录调控奠定了基础。     

幽门螺杆菌诱导胃上皮细胞白细胞介素-8基因转录的信号转导机制

背景:亚洲与欧美国家的幽门螺杆菌(H.pylori)细胞毒素相关基因致病岛(cogPAI)存在地域差异,但此种差异与胃上皮细胞白细胞介素(IL)-8基因转录信号转导机制之间的关系尚不明确.目的:探讨我国H.pylori菌株诱导胃上皮细胞IL-8基因转录的信号转导机制.方法:以DNA杂交法分析长海医院5株临床分离H.pylori菌株的cagPAI状态.将含IL-8报告基因的人胃上皮细胞系L5F11分别与5株H.pylori共培养,或与各蛋白激酶(PK)抑制剂预作用1 h后再行共培养,用液体闪烁计数仪测定L5H11细胞的荧光素酶活性(IL-8基因转录),用酶联免疫吸附测定(ELISA)检测IL-8蛋白浓度.结果:在所分离的5株临床H.pylori菌株中,4株具有完整的cag PAI,1株cag PAI部分丢失.蛋白酪氨酸激酶(PTK)抑制剂除莠霉素A不仅能显著抑制H.pylori诱导的L5F11细胞的荧光素酶活性(P＜0.05或P＜0.01),而且能抑制其IL-8蛋白的分泌(P＜0.05或P＜0.01);PKG抑制剂KT5823和PKC抑制剂抑激酶素C对H.pylori诱导的IL-8基因转录和蛋白分泌均无影响.结论:与欧美国家的H.pylori菌株一样,本研究临床分离的H.pylori菌株亦通过PTK途径诱导胃上皮细胞IL-8基因转录.     

AP-2α对基质金属蛋白酶-20作用的研究

目的:通过研究AP-2α转录因子对基质金属蛋白酶-20(matrix metalloproteinase-20,MMP-20)基因表达的调控作用,进一步明确AP-2α的功能,为研究AP-2α在釉质形成中的作用奠定基础。方法:利用RT-PCR法、双荧光素酶报告基因检测系统、染色体免疫共沉淀技术等来分析AP-2α与MMP-20启动子区域的转录调控能力。结果:小鼠成釉细胞转染AP-2αsiRNA后,成釉细胞中MMP-20 mRNA的表达水平显著上调。染色体免疫共沉淀结果显示,AP-2α可能通过与MMP-20启动子特征性序列相互作用,从而调控MMP-20的表达。双荧光素酶报告基因检测显示预测结合位点突变后,AP-2α对MMP-20启动子的表达水平无显著影响。结论:AP-2α基因沉默可显著上调MMP-20的mRNA表达水平;而AP-2α过表达可显著抑制MMP-20活性。提示AP-2α转录因子与成釉细胞内MMP-20启动子的特征性序列相互作用,从而调控MMP-20的表达水平。     

变形链球菌荧光报告株的构建和鉴定

目的探讨变形链球菌(S.mutans)荧光素酶(luc)基因报告株构建方法,拟为探讨抗菌剂对多菌种生物膜中S.mutans的作用提供研究基础。方法将S.mutans UA159乳酸脱氢酶(ldh)基因及其上游部分约1100000片段克隆至自杀质粒pFW5-luc的多克隆位点,构建重组质粒,并经酶切和测序证实。采用自然转化的方法,实现重组自杀质粒和S.mutans UA159同源序列的单次交换。结果经过聚合酶链反应(PCR)和测序分析,筛选出具有报告活性的S.mutans ldh-luc基因荧光报告株。结论成功构建了S.mutans ldh-luc基因荧光报告株。     

人MicroRNA-335阳性对照质粒真核表达载体的构建及意义

目的构建人MicroRNA-335(has-miR-335)阳性对照质粒表达载体。方法根据与has-miR-335"种子区"完全匹配的序列设计并合成一对互补的寡聚核苷酸链,经过缓慢降温退火得到目的小片段。将退火产物连接入荧光素酶报告基因载体p MIR-REPORT而得到has-miR-335阳性对照重组质粒。应用Lipofectamine 2000将has-miR-335阳性对照重组质粒、p MIR-REPORT空载体,分别与Pre-miRTMmicroRNA335 Precursor共转染至293T7/17细胞,用双荧光素酶方法检测has-miR-335在真核细胞中表达水平。结果获得has-miR-335阳性对照重组质粒,经测序结果正确。has-miR-335与其阳性对照结合在真核细胞中的表达下降。结论成功构建了has-miR-335阳性对照质粒表达载体。该载体可用于确认has-miR-335实验中RNA提取、转染和基因表达检测方法是否可靠。has-miR-335双荧光素酶报告基因检测体系的建立为进一步进行has-miR-335靶基因检测及验证奠定基础。     

PLUNC基因启动子区荧光素酶报告载体的构建与鉴定

目的构建人PLUNC（palate,lung and nasal epithelium clone）基因启动子区C-1888T SNP位点不同单倍型荧光素酶报告基因表达载体。方法利用PCR技术得到1888位点不同基因型的PLUNC基因启动子区目的片段．将其分别插入到pMD18-T载体和pGL3-enhancer荧光素酶报告基因载体中并测序。结果成功构建了人类PLUNC基因C-1888T SNP位点上的2种不同纯合子基因型（CC型和TT型）的荧光素酶报告载体（pGL3-enhancer—T型和pGL3-enhancer—C型）,且测序得以证实。结论成功构建出的荧光素酶报告载体,为研究PLUNC基因不同的1888SNF位点能否调控不同的PLUNC基因表达提供了基本实验条件。     

人SREBP-1c启动子报告基因的构建和功能验证

目的 构建人SREBP-1c启动子的荧光素酶报告基因载体并进行功能的初步验证。 方法 提取人血基因组DNA,PCR扩增SREBP-1c的启动子,构建SREBP-1c启动子双荧光素酶的报告基因载体;用双荧光素酶活性检测其功能。Western blotting检测FoxO1对SREBP-1c蛋白的抑制作用。 结果 成功构建了pGL3-Basic-SREBP-1c-promoter的报告基因载体,共转染FoxO1明显抑制了人SREBP-1c启动子的活性,同时过表达FoxO1抑制了SREBP-1c的蛋白表达。 结论 FoxO1通过抑制SREBP-1c的转录来抑制SREBP-1c的表达。     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.