大鼠GluR2基因启动子控制的萤火虫荧光素酶表达载体的构建

目的 构建大鼠GluR2基因启动子控制的萤火虫荧光素酶表达载体,并检测其在神经元中的表达.方法 采用PCR方法获得GluR2基因启动子区目的 片段(-298～﹢283),双酶切后插入到pGL3-Basic载体中构成重组表达载体,使萤火虫荧光素酶报告基因的表达受GluR2启动子控制.将构建的重组表达载体或pGL3-Basic载体分别与内参质粒pRL-CMV(表达海肾荧光素酶)共转染原代培养皮质神经元,24 h后用双荧光检测试剂盒测定萤火虫荧光素酶及海肾荧光素酶活性.结果 重组表达载体经双酶切及测序鉴定证明构建正确,该重组表达载体在神经元中特异性高表达萤火虫荧光素酶.结论 成功构建大鼠GluR2基因启动子控制的萤火虫荧光素酶表达载体,并检测到该载体在神经元中的特异性表达.     

复制子荧光素酶表达质粒pSVK-luc的构建与应用

目的 为研究复制子DNA疫苗在生物活体内的表达情况,构建含有荧光素酶报告基因的复制子表达质粒pSVK-luc.方法 PCR扩增荧光素酶报告基因,克隆入DNA复制子载体pSVK,酶切鉴定和测序分析筛选阳性重组质粒pSVK-luc;化学法转染人胚肾293T细胞,流式细胞术和免疫荧光法检测荧光素酶基因在293T细胞中的表达;利用基因电穿孔导入仪递送重组质粒至BALB/c小鼠股四头肌,活体成像仪动态观测荧光素酶基因在小鼠体内的表达.结果 pSVK-luc经相应酶切和测序鉴定,与预期设计完全一致.流式细胞术和免疫荧光检测均可见荧光素酶基因表达;同时活体成像仪也能检测到该基因在小鼠体内的表达.结论 pSVK-luc表达质粒的构建成功将为复制子DNA疫苗体内作用机制研究和体内电穿孔递送条件的优化奠定实验基础.     

含有增强型绿色荧光蛋白报告基因的辛德毕斯病毒的制备与鉴定

目的 制备含有增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的辛德毕斯病毒,并对其进行鉴定.方法 采用融合PCR技术将报告基因EGFP融合到辛德毕斯病毒XJ-160感染性克隆pBR-XJ160的结构基因编码框后,并在报告基因前面添加亚基因启动子SP6,通过反向遗传学技术拯救得到EGFP标记的辛德毕斯病毒.结果 成功拯救并获得了EGFP标记的XJ-160病毒,该重组病毒具有良好的荧光表达特性和遗传稳定性.结论 EGFP标记的辛德毕斯病毒可用于观测病毒的侵染轨迹,为进一步研究辛德毕斯病毒嗜性、生物学功能,以及感染机制奠定了基础.     

人GDDR基因启动子的克隆和报告基因载体构建

目的:克隆人GDDR基因的启动子;构建GDDR启动子的报告基因载体并进行活性分析.方法:设计合成GD-DR启动子引物,采用PCR技术从人基因组DNA中扩增GD-DR启动子;将扩增片段插入T载体并利用酶切与测序进行鉴定;亚克隆该基因至pGL3-Basic荧光报告基因载体;瞬时转染细胞,用双荧光素酶报告基因系统检测报告基因载体的活性.结果:PCR扩增得到人GDDR基因启动子;成功构建pGL3-GDDR-promoter 报告基因载体,测序结果表明启动子序列正确;双荧光素酶报告基因检测系统证实构建的报告基因载体具有启动子活性.结论:成功地构建人GDDR基因启动子的克隆及其报告基因载体的构建,为深入研究GDDR转录表达的调控机制提供基础.     

人IFN-β基因启动子荧光表达载体的构建及鉴定

目的构建人IFN-β基因启动子萤火虫荧光素酶报告基因载体(pGL3-IFNB1)及增强型绿色荧光蛋白(EGFP)表达载体(pGE3-IFNB1),并在A549细胞中进行表达验证。方法采用PCR方法扩增出IFN-β启动子区片段,克隆入荧光表达载体pGL3 basic和pGE3 basic,并用脂质体瞬时转染A549细胞,6 h后用汉滩病毒(HTNV)感染转染后的细胞,24 h后检测重组载体在细胞内的表达情况。结果重组载体pGL3-IFNB1和pGE3-IFNB1分别经双酶切鉴定及序列测定证实载体构建正确;且在A549细胞内成功表达。经HTNV刺激后表达明显增加。结论成功构建了人IFN-β启动子荧光素酶报告基因载体(pGL3-IFNB1)及增强型绿色荧光蛋白表达载体(pGE3-IFNB1),为进一步研究病毒诱导产生IFN-β机制提供了有用的工具。     

小鼠CXCR4基因启动子的克隆和报告基因载体构建

目的:克隆小鼠CXCR4基因启动子, 并建立CXCR4报告基因系统.方法:设计合成PCR引物, 从小鼠基因组DNA中扩增并克隆小鼠CXCR4基因的启动子区.序列测定确认后, 用克隆的启动子片段构建CXCR4荧光素酶报告基因载体pGL-CXCR4.转染细胞, 用双报告基因系统检测报告基因载体的活性.结果:成功扩增了小鼠CXCR4基因的启动子区.克隆入质粒载体后经DNA序列测定证实了其序列.通过转染细胞和荧光素酶分析, 证实所构建的报告基因可以反映CXCR4启动子的活性.结论:成功扩增、克隆了小鼠CXCR4基因启动子, 并成功建立起其报告基因系统, 为后续的研究奠定了基础.     

阴道毛滴虫LAG1基因启动子区克隆及分析

本研究旨在克隆并分析阴道毛滴虫LAG1基因启动子,为进一步研究在细胞衰老过程中LAG1基因的转录调控提供实验资料。预测启动子所在区域,用PCR技术扩增启动子区序列,并分别克隆入荧光素酶报告基因载体pGL3-Basic及增强型绿色荧光蛋白报告基因载体pEGFP-1,用脂质体介导的方法瞬时转染EC109细胞,然后测定荧光素酶表达活性及观察绿色荧光蛋白的表达情况。结果表明在构建的6种荧光素酶报告基因表达体系及2种增强型绿色荧光蛋白报告基因表达体系中,表达载体pGL3-455(-455~ 55bp)、pGL3-417(-417~ 55bp)、pGL3-280(-280~ 55bp)、pGL3-202(-202~ 55bp)、pGL3-81(-81~ 55bp)的荧光素酶表达活性相近,均明显高于表达载体pGL3-47(-47~ 55bp)荧光素酶表达活性,而pGL3-47表达载体荧光素酶表达活性极低,与阴性对照活性相近,提示-47~ 55bp区无启动子活性。绿色荧光蛋白的表达情况也类似,pEGFP-81(-81~ 55bp)有明显的绿色荧光蛋白表达,而pEGFP-47(-47~ 55bp)和空载体pEGFP-1未见表达。因此-81~-47bp区域含有阴道毛滴虫LAG1基因转录所必需的基本启动子序列。     

SPA基因启动子的克隆及转录靶向活性分析

目的：克隆大鼠肺表面活性蛋白A（SPA）基因启动子并构建该启动子的荧光报告系统,探讨该启动子的活性及转录靶向性，为进一步研究SPA 基因的表达调控机制和探讨靶向性基因治疗奠定基础。方法:①从GenBank中获取大鼠SPA 基因序列，对其上游基因组序列进行计算机生物信息学分析，推断其上游序列约163bp的区域具有启动子功能。②利用PCR技术扩增SPA 基因上游启动子序列,将其亚克隆入pGL3-basic 中,构建pGL3-SPA 质粒，将其亚克隆于pGL3-control 中，构建pGL3-SPA-enhancer 质粒。③将构建pGL3-SPA质粒、pGL3-SPA-enhancer 质粒、pGL3-control质粒和pGL3-basic 质粒分别与内参质粒pRL-TK共转染入A549细胞和H441细胞, 用双荧光素酶报告系统检测该质粒在两种细胞中的荧光素酶活性表达。 结果:酶切及测序结果均证实成功克隆了SPA基因启动子序列,并已将该序列正确插入到荧光素酶报告基因系列载体中。重组的pGL3-SPA-enhancer 质粒、pGL3-SPA质粒转染H441 细胞后可以检测到荧光素酶的高表达。结论:成功构建了含有SPA 基因启动子序列的荧光素酶基因报告系统,证实了它在高表达SPA蛋白的细胞中有较高的转录活性， 为下一步研究SPA 基因功能及其转录调控以及探讨基因治疗的靶向性奠定了基础。     

含人P21启动子双荧光素酶基因载体的构建

目的构建带有EGFP标记含人P21启动子的双荧光素酶报告基因载体。方法从人血白细胞DNA中克隆P21启动子,用于控制firefly荧光素酶的表达;renilla荧光素酶基因和EGFP基因用IRES连接,在CMV启动子控制下表达;上述元件和基因克隆至载体pLL3.7中,应用酶切和测序的方法进行鉴定正确后转染至293FT细胞观察EGFP表达。结果经酶切、测序证实成功构建了带有EGFP标记的含人P21启动子的双荧光素酶报告基因的载体,并成功表达EGFP。结论含人P21启动子的双荧光素酶报告基因的载体成功构建,为下一步的药物筛选打下基础。     

癌胚抗原启动子控制基因靶向表达研究

目的 构建由癌胚抗原(CEA)启动子控制报道基因增强型绿色荧光蛋白(EGFP)表达的重组表达质粒CMVE-pCEA-IRES-EGFP.了解CEA启动子靶向驱动的含目的基因的重组质粒的效率.方法 构建重组质粒CMVE-pCEA-IRES-EGFP,以PCR,DNA测序及酶切进行鉴定.通过脂质体介导重组质粒CMVE-pCEA-IRES-EGFP体外分别转染A549细胞(CEA阳性)和16HBE(CEA阴性)细胞,进行绿色荧光蛋白检测分析基因表达的靶向性.结果 CMVE-pCEA-IRES-EGFP分别用VSP Ⅰ,VSP Ⅰ/Nhe Ⅰ酶切后电泳分别出现大小约405 bp,372 bp和4110 bp的片断,与预计各片断大小相符.以CMVE-pCEA-IRES-EGFP为模板PCR扩增得到CMV和CEA片段,分别连于T载体(pMD18-T)并测序,结果正确.嵌合启动子CMVE-pCEA能特异地驱动下游报告基因在CEA阳性肺癌细胞株绿色荧光表达阳性,而CEA阴性细胞株16HBE表达阴性.结论 成功构建了嵌合启动子CMVE-pCEA驱动的CMVE-pCEA-IRES-EGFP重组质粒,并能在转染的CEA阳性细胞株中表达,为靶向启动双基因表达载体构件提供了实验依据.     

HRE/CMV启动子的克隆和缺氧调控活性的测定

目的： 将具有缺氧调控作用的多种缺氧反应元件 (HRE)与CMV启动子组合，以获得缺氧应答启动子。将荧光素酶报告基因置于缺氧应答启动子的下游，通过检测荧光素酶活性的方法，方便地研究缺氧应答启动子缺氧诱导的作用。方法: 合成多种HRE核苷酸序列，并设立HRE突变对照，克隆入pCI-neo中，构建HRE/CMV启动子。扩增HRE/CMV序列，定向亚克隆入pGL3-Basic中，获得HRE/CMV荧光素酶报告载体。瞬时转染HeLa细胞，在0.1% O₂ 环境下培养细胞，并设立正常氧分压环境对照。检测荧光素酶的相对活性。结果: 成功构建了HRE/CMV荧光素酶报告载体，其中mPGK-HRE/CMV载体表现出很好的缺氧诱导作用和高水平的表达。结论: mPGK-HRE/CMV启动子能够有效地进行缺氧诱导和调控工作。     

利用合适的启动子提高外源基因在Jurkat细胞中的表达

目的通过选择合适的启动子来实现外源基因在Jurkat细胞中的高效表达。方法以pGL3-MCS质粒为基础构建不同启动子(SV40、CMV、EF1α和UbC)驱动萤火虫荧光素酶Fluc报告基因表达的重组质粒,并将其与作为内参的表达海肾荧光素酶Rluc报告基因的质粒共转Jurkat细胞,检测Jurkat细胞中这两种荧光素酶与相应底物反应所产生的荧光强度,计算Fluc与Rluc荧光强度的比值即Fluc的相对酶活,通过比较Fluc相对酶活的大小来选取在Jurkat细胞中具有高表达活性的启动子。结果以pGL3-MCS质粒为基础成功构建了不同启动子驱动Fluc报告基因表达的重组质粒pGL3-CMV、pGL3-EF1α和pGL3-UbC。在Jurkat细胞中,不同启动了驱动的Fluc报告基因的相对酶活的强弱关系为pGL3-UbCpGL3-EF1αpGL3-CMVpGL3-MCS。结论可以选择高活性的人源泛素C启动子实现外源基因在Jurkat细胞中的高效表达,以利于Jurkat细胞系在哺乳动物T淋巴细胞以及免疫相关功能研究中的应用。     

活化T细胞核因子对组成性启动子SV40的调控作用

目的:利用双荧光素酶报告系统探讨活化T细胞核因子(NFAT)能否调控常见组成性启动子CMV,SV40和TK,为不同条件下选择合适双荧光报告系统内参提供依据。方法:用限制性内切酶BglⅡ和HindⅢ分别从质粒pCDNA3.1和pRL-TK中切下CMV和TK启动子,克隆至pGL3-basic载体中,构建成pCMV-Luc和pTK-Luc载体。将构建pCMV-Luc和pTK-Luc以及商品化的pGL3-control(SV40启动子驱动),分别与SV40(pBIND)和TK(pRL-TK)两种启动子驱动的两种内参质粒共转染入HEK293细胞;观察过表达组成性活化NFAT后相对荧光素酶活性读数的改变。结果:成功构建了pCMV-Luc和pTK-Luc质粒,荧光素酶活性检测发现,常见组成性启动子SV40启动子对过表达组成性活化的NFAT存在一定的反应。结论:T细胞活化过程中重要的转录因子NFAT能够调控SV40启动子活性;表明常见组成性启动子SV40并非真正、绝对的组成性不变。因此,在荧光素酶报告系统内参选择时需要充分考虑该问题,本研究为合理选择内参质粒提供了一个可行策略。     

Induction of humoral and cellular immune responses against hepatitis C virus by vaccination with replicon particles derived from Sindbis-like virus XJ-160

A replication-defective, recombinant Sindbis virus vector was utilized in a novel immunization strategy to induce humoral and cellular responses against hepatitis C virus (HCV). The recombinant vector, pVaXJ-E1E2, expressing the gene for HCV glycoproteins E2 and E1, was constructed by inserting the E1E2 gene into the replicon pVaXJ, a DNA vector derived from Sindbis-like virus XJ-160. The defective replicon particles, XJ-E1E2, were produced by transfecting BHK-21^E+Capsid cells, the packaging cell lines for the vector from XJ-160 virus, with pVaXJ-E1E2. Both glycoproteins, E2 and E1, were stably expressed, as indicated by immunofluorescence assay (IFA) and Western blotting. Mice were vaccinated using a prime-boost strategy with XJ-E1E2 particles combined with Freund’s incomplete adjuvant via intramuscular injection at 0 and 2 weeks. HCV-specific IgG antibody levels and cellular immune responses were evaluated by IFA and IFN-γ ELISPOT, respectively. The results showed that the defective XJ-E1E2 particles in combination with Freund’s incomplete adjuvant induced effective humoral and cellular immune responses against HCV glycoprotein E1 or E2, suggesting that a defective Sindbis particle vaccine is capable of eliciting an effective immune response. These findings have important implications for the development of HCV vaccine candidates.     

Noncytopathic flavivirus replicon RNA-based system for expression and delivery of heterologous genes

Noncytopathic replicons of the flavivirus Kunjin (KUN) were employed for expression and delivery of heterologous genes. Replicon vector C20DX2Arep, containing a unique cloning site followed by the sequence of 2A autoprotease of foot-and-mouth disease virus, was constructed and used for expression of a number of heterologous genes including chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), beta-galactosidase, glycoprotein G of vesicular stomatitis virus, and the Core and NS3 genes of hepatitis C virus. The expression and proper processing of these genes upon transfection of BHK21 cells with the recombinant replicon RNAs were demonstrated by immunofluorescence, radioimmunoprecipitation, and appropriate reporter gene assays. Most of these recombinant KUN replicon RNAs were also successfully packaged into secreted virus-like particles (VLPs) by subsequent transfection with Semliki Forest virus replicon RNA expressing KUN structural genes. Infection of BHK21 and Vero cells with these VLPs resulted in continuous replication of the recombinant replicon RNAs and prolonged expression of the cloned genes without any cytopathic effect. We also developed a replicon vector for generation of stable cell lines continuously expressing heterologous genes by inserting an encephalomyelocarditis virus internal ribosomal entry site-neomycin transferase gene cassette into the 3'-untranslated region of the C20DX2Arep vector. Using this vector (C20DX2ArepNeo), stable BHK cell lines persistently expressing GFP and CAT genes for up to 17 passages were established. Thus noncytopathic KUN replicon vectors with the ability to be packaged into VLPs should provide a useful tool for the development of noninfectious and noncytopathic vaccines as well as for gene therapy applications.     

HER2启动子在卵巢癌细胞系中控制报告基因特异性表达

目的 构建一种能在卵巢癌肿瘤组织中进行肿瘤特异性表达的逆转录病毒载体,增强肿瘤基因治疗的靶向性。方法 ＰＣＲ法获得ＨＥＲ２基因的启动子片段并将其克隆至逆转录病毒载体Ｐ＾ＬＸＳＮ中,再将突变型绿色荧光蛋白（ｍｔＧＦＰ）基因克隆至该启动子下游,将此载体转染ＳＫ－ＯＶ３细胞和ＭＣＦ－７细胞。结果 转染３天后可见部分ＳＫ－ＯＶ３细胞呈现绿色荧光,而ＭＣＦ－７中无此现象。结论 ＨＥＲ－２基因启动子可控制报告     

A novel adenoviral vector-mediated neuronal selective gene expression in neonatal mouse brain in response to hypoxia

Selective gene expression targeting neurons is a challenge, which, if successfully overcome, carries an enormous potential for clinical applications in therapeutics against neurodegenerative diseases. We have reported previously the construction of a series of adenoviral vectors capable of selectively expressing a reporter gene luciferase in cultured neurons [D. Huang, A. Desbois, S.T. Hou, A novel adenoviral vector which mediates hypoxia-inducible gene expression selectively in neurons, Gene Ther. 12 (2005) 1369-1376]. A combination of neuron restrictive silencer elements (NRSEs), hypoxia responsive elements (HREs) and CMV minimal promoter (CMVmp) was packaged into replication defective adenovirus to target gene expression selectively in neurons in a hypoxia-regulated manner. In the present study, we injected the adenoviral vectors into the neonatal mouse brain followed by treatment with hypoxia. The expression of the reporter luciferase gene was examined by luciferase assay and fluorescent immunostaining. Neurons and glial cells were identified by staining with antibodies against NeuN and GFAP, respectively. Remarkably, in response to hypoxia, Ad/5HRE-3NRSE showed strong hypoxia-inducible gene expression of the reporter luciferase selectively in neurons but not in glial cells. In contrast, brains infected with the control vector Ad/5HRE showed no selectivity in luciferase expression (in both neurons and glial cells) under the hypoxic condition. Taken together, these studies demonstrated that this vector (Ad/5HRE-3NRSE) can mediate gene expression selectively in neurons both in vitro and in vivo, supporting the suggestion that further refinement of this vector may lead to the development of a useful tool to investigate mechanisms of neuronal damage following cerebral ischemia and a possible effective gene therapy vector to stroke.