EphA3基因启动子区域定位表达及活性分析

目的构建含有不同长度EphA3基因启动子片段的报告基因载体,研究其在293T细胞和MEF细胞中的转录活性。方法以Balb／C小鼠基因组DNA为模板,扩增不同长度的EphA3基因启动子片段,并克隆进入荧光素酶报告基因质粒pGL3-Basic真核表达载体内。酶切鉴定及基因测序无误后,将重组质粒和pRL—CMV内对照质粒共转染293T和MEF细胞,分析不同长度的OhA3基因启动子片段的转录活性。结果酶切和测序鉴定表明表达载体构建成功,EphA3基因的核心启动子区域位于-279bp～＋110bp之间,在293T细胞和MEF细胞中其转录活性相似。结论成功构建了荧光素报告基因重组质粒,并确定了BphA3基因的核心启动子区域。     

基于双质粒拯救系统的新城疫病毒LaSota株的拯救

传统新城疫病毒(newcastle disease virus, NDV)的拯救系统包括一个cDNA克隆质粒和分别表达NDV的核衣壳蛋白(NP)、磷蛋白(P)、聚合酶蛋白(L)的3个辅助质粒,且必须满足4个质粒同时转染进入同一个宿主细胞才能完成病毒的组装,效率相对低下。【目的】提高NDV的拯救效率,并建立双质粒高效拯救系统。【方法】将NP、P、L基因表达盒串联克隆至真核表达载体pCI中,构建为可同时表达NP、P、L蛋白的单辅助质粒PCI-NPL;同时,采用分段克隆再拼接的方式,将NDV LaSota株基因组cDNA克隆于真核表达质粒pCI的CMV启动子下游,并分别在P和M基因中插入报告基因增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP)、5''端引入锤头状核酶序列、3''端引入丁型肝炎病毒核酶序列,构成全基因组转录质粒pCI-LaSota-EGFP;以pCI-LaSota-EGFP和pCI-NPL组成病毒拯救系统共转染至BHK-21细胞,拯救获得重组子代病毒rLaSota-EGFP,并进行系列生物学特性鉴定。【结果】经RT-PCR、荧光显微镜观察、Western blotting、生长特性测定等系列鉴定,证明rLaSota-EGFP构建正确,成功拯救获得了重组病毒rLaSota-EGFP,且与野生型(wild-type, WT) LaSota具有相似的生物学特性。【结论】基于CMV启动子的NDV双质粒新型拯救系统构建成功,为重组NDV及其他副黏病毒的高效拯救奠定了基础。     

犬瘟热病毒小熊猫株反向遗传系统的构建及其鉴定

以驯化致弱的犬瘟热病毒小熊猫株(Canine distemper virus,CDV)为模板,构建犬痘热病毒感染性cDNA克隆.对其全基因组序列测定后,用RT-PCR的方法获得组成全长基因的7个片段,通过酶切、拼接将7段CDVcDNA序列插入到真核表达载体pCI的MCS上,构建犬瘟热病毒小熊猫株的全长cDNA质粒(pCI-CDV-LP),同时分别克隆CDV小熊猫株N、P、L蛋白ORF构建三个辅助质粒.酶切鉴定和序列测定表明,pCI载体中插入的核酶及CDV cDNA序列正确无误,使用转染试剂Lipofectamine TM 2000将全长质粒和三个辅助质粒共转染中国仓鼠肾细胞(BSR),经RT-PCR、间接免疫荧光和病毒感染VERO-SLAM细胞试验鉴定,成功拯救出CDV小熊猫株,显示CDV小熊猫株反向遗传系统构建完成,为犬瘟热病毒致病机理及免疫研究奠定基础.     

含G0S2基因启动子的荧光素酶报告基因载体的构建

目的:克隆人G0S2基因启动子并构建荧光素酶报告基因载体,为进一步研究G0S2基因转录调控提供质粒。方法:利用PCR技术从人胚肾293A细胞基因组DNA中克隆获得G0S2基因启动子的DNA片段,将其克隆至pGL3-basic表达载体中,并转化人大肠杆菌DH5α,经限制性内切酶酶切、PCR及测序鉴定得到确认;将重组载体质粒与半乳糖苷酶表达质粒psV-β-Galactosidase共转染至大鼠血管平滑肌细胞(VSMC),检测细胞中荧光素酶的活性。结果:pGL3-G0S2-Promoter重组质粒插入片段和相邻序列正确,克隆的G0S2基因片段有启动子活性(P0.05)。结论:成功构建了pGL3-G0S2-Promoter报告基因质粒,为进一步研究G0S2基因的表达奠定了基础。     

ZNF268基因启动子区的克隆及功能分析

锌指基因家族是人体中最大的基因家族,它参与细胞分化、胚胎发育,并与许多疾病的发生相关。人类ZNF268基因是一个在人胚肝中特异性表达的C2H2型锌指基因,并可能在人的早期肝脏发育中起重要作用。为了研究ZNF268基因表达调控的分子机制,以正常人总基因组为模板PCR扩增了ZNF268基因的5′调控区2533bp片段,并将此片段插入启动子缺失的EGFP(增强型绿色荧光蛋白)载体构建了重组质粒pZNF268—EGFP。用脂质体介导的方法将pZNF268—EGFP转染NIH／3T3、COS7、K562、HeLa4个细胞系。在激光共聚焦显微镜下观察绿色荧光的表达,发现在每个细胞系中,转染了重组质粒pZNF268—EGFP的细胞均有荧光表达,但其起始表达时间均晚于转染了阳性对照质粒pEGFP—C1的细胞且荧光较弱。这表明ZNF268基因的5′调控区2．5kb片段是一个有功能的启动子,但该启动子与CMV启动子相比活性较弱。选择易培养的HeLa细胞系用于缺失研究。将一系列5′端—2456bp至—20bp缺失、3′端均为 77bp的缺失片段插入启动子缺失的CAT(氯酶素酰胺转移酶)载体构建了一系列重组质粒。将这些重组质粒转染HeLa细胞系进行缺失分析,并通过共转染pCMV—Sport—βgal质粒校正转染效率。结果表明,ZNF268启动子—2456～—1639bp区域可能含有正调控元件,—1244～—1013bp和—525～—156bp区域可能含有负调控元件,ZNF268启动子激活转录的一个重要区域位于—156～—20bp。     

丙型肝炎病毒NS2蛋白在Huh-7细胞中抑制NF-κB活性

将丙型肝炎病毒(HCV)非结构蛋白NS2基因的全长序列插入到真核表达载体pCDNA3.1(-)CMV启动子下游,构建成重组质粒pCNS2.用脂质体LipoVecTM转染Huh-7细胞,转染细胞内可检出NS2的mRNA和蛋白质,表明构建的pCNS2可在Huh-7细胞内成功表达;把不同剂量的pCNS2质粒DNA与报告质粒pNF-κ B-Luc共转染Huh-7细胞,48h后检测荧光素酶活性,结果显示与pCNS2共转染的细胞中pNF-κ B-Luc表达出的荧光素酶的活性比对照细胞降低了约2～4倍,并呈明显的剂量相关性.表明HCV NS2对NF-κ B激活转录活性有明显的抑制作用.这可能与HCV慢性持续性感染的致病性有一定的相关性.     

麻疹病毒沪191株反向遗传系统的建立

建立麻疹病毒沪191株(MV-_(S191))反向遗传系统。提取MV-_(S191)的RNA,RT-PCR分7段扩增出麻疹病毒反基因组cDNA,通过酶切、连接,拼接出MV-_(S191)全长反基因组,并分别在其3′端和5′端引入丁型肝炎病毒核酶序列(HdvRZ)和锤头核酸酶序列(HamRZ),克隆到pT7-MCS载体,获得pT7MV_(S191)。在MV-_(S191)的P-M基因间区域插入绿色荧光蛋白(GFP),获得pT7MV_(S191)-ATU-GFP,同时构建表达麻疹病毒核蛋白(N)、磷蛋白(P)和大蛋白(L)的3个辅助质粒。将pT7MV_(S191)、pT7MV_(S191)-ATU-GFP分别与辅助质粒通过脂质体介导共转染BSRT7细胞,热休克3h。转染3d后,取上清,感染Vero-SLAM细胞。经RT-PCR、显微镜检测绿色荧光蛋白表达及合胞体形成,在该系统中成功拯救到两种有感染活性的病毒。成功建立了MV-_(S191)的反向遗传系统,为以麻疹病毒为载体进行新疫苗的研发奠定了基础。     

T7启动子在哺乳类动物细胞中启动外源基因表达的研究

人低密度脂蛋白（LDL）受体基因cDNA和氯霉素已酞转移酶基因（CAT）及PolyA信号序列被克隆进pGEM4载体的T7噬茵体启动子下游,构建成质粒pT7LDLR和pT7CAT．两个重组质粒转化CHO细胞．PCR和CAT酶实验显示：两个基因被T7噬菌体启动子所启动．结果证实真核生物RNA聚合酶能够识别T7启动子,转录外源基因．常用的含有T7启动子的质粒可同时作为原核生物和真核生物的表达载体．     

准噶尔雅罗鱼β-肌动蛋白基因启动子的克隆及其活性功能初步检测

以开发利用新疆濒危鱼类准噶尔雅罗鱼(Leuciscus merzbacheri)基因资源为研究目的, 利用PCR技术克隆准噶尔雅罗鱼的β-actin 基因, 得到的β-actin 基因片段SZ21包含启动调控区, 大小为2 398 bp。SZ21的启动调控区包括β-actin 基因上游调控序列、第1、第2、第3和第4外显子部分序列。上游调控序列中含有对转录起重要作用的CAAT框、TATA 框和CArG 框等元件。对启动子序列在线分析表明, 获得的启动子含有E-box、RU49、ZBPF、CEBP、CREB等多个重要转录因子结合位点。用AatⅡ破坏真核表达载体pEGFP-N1-AFPⅢ中的CMV启动子, 将准噶尔雅罗鱼的SZ21启动调控区克隆到载体pEGFP-N1-AFPⅢ(CMV坏)上, 构建成重组表达载体β2 pEGFP-N1-AFPⅢ。脂质体转染BHK-21细胞。结果表明, 克隆的准噶尔雅罗鱼β-actin基因启动子SZ21具有启动EGFP报告基因在哺乳动物细胞中表达的活性。通过BHK-21绿色荧光细胞的传代证实, 克隆的启动子具有持续启动蛋白基因表达的活性, 在细胞传代中可以遗传。PCR检测传代的BHK-21绿色荧光细胞基因组DNA, 均能检测到SZ21目的片段。文章成功分离了具有活性功能的准噶尔雅罗鱼β-actin基因启动子。     

仙台病毒BB1株6个编码基因的克隆及表达

在仙台病毒BB1株全基因组序列测定的基础上,用反转录和PCR方法获得了核蛋白基因(N),磷蛋白基因(P),神经血凝素基因(HN),基质蛋白基因(M)、融合蛋白基因(F)和聚合酶蛋白基因(L)等6个编码基因全长克隆;测序结果表明,其序列与Genbank中登录的序列(DQ219803)完全一致。为了提供仙台病毒基因组载体拯救和包装所需的反式作用蛋白,将N、P、M、F、HN和L分别克隆到腺病毒穿梭表达载体pDC316上,将它们分别与腺病毒基因组质粒pBHGlox△E1,3Cre共转染HEK293细胞,获得了6种复制缺陷性重组腺病毒Ad5-N、Ad5-P、Ad5-M、Ad5-F、Ad5-HN和Ad5-L。酶切结果表明6种重组腺病毒穿梭质粒构建正确;用PCR方法证明所获得的6种重组腺病毒分别携带了上述6个编码基因;用重组腺病毒感染LLC-MK2细胞后用Western blotting和免疫荧光方法检测到了相应仙台病毒编码基因的表达。本研究为仙台病毒BB1株全长基因组的拼接和病毒载体包装系统组建打下了基础。     

Cytoplasmic expression of ribozyme in zebrafish using a T7 autogene system

A cytoplasmic ribozyme expression system, based on codelivery of a ribozyme vector, a T7 autogene vector, and T7 RNA polymerase (RNAP), has been developed and used to generate a specific phenotype in zebrafish by targeting a no tail (ntl) mRNA. The expression of the no tail ribozyme sequence is under the control of a tandem of two promoters: The T7 promoter and an adenoviral va 1 (pol III) promoter. The coinjection of the ribozyme vector pT7vaRz, the T7 autogene vector pT7T7, and the T7 RNAP resulted in rapid synthesis of the ribozyme against the ntl mRNA in the cytoplasm of the injected zebrafish embryos, generating no tail phenotypes in up to 10-20% of the injected embryos. The phenotypic change rates have been found to be related to the concentrations of the plasmid vectors and T7 RNAP injected and to the ratios of the three injected components. This cytoplasmic ribozyme expression system may be useful for efficiently targeting other mRNA and for various biomedical applications. These potential applications may include rapid identification of biological functions of novel genes from zebrafish and humans based on partial gene sequence information and gene therapy of genetic and acquired diseases.     

原核表达系统T7RNA聚合酶/启动子在真核细胞中表达目的基因的实验研究

将目前高表达水平强大的原核表达系统之一T7 RNA聚合酶/启动子表达系统通过一系列改进引入真核细胞.通过转染真核细胞实验表明,采用真核启动子CMV调控T7 RNA聚合酶的表达和在T7启动子下游插入EMCV IRES序列两种解决方案能使该原核表达系统在真核细胞高效表达目的基因,且能适应不同的真核细胞环境,是一良好的细胞类型非依赖的表达体系.     

用表达T7RNA聚合酶细胞系拯救麻疹病毒微复制子

构建稳定表达T7RNA聚合酶的细胞系,用于提高拯救麻疹病毒微复制子效率.PCR扩增T7RNA聚合酶基因,克隆到真核表达载体,转染Vero细胞,用G418筛选到稳定表达的细胞株Vero/pcDNA3-T7.用Westernblotting证明了T7RNA聚合酶在细胞中的表达.将T7启动子控制绿色荧光蛋白表达的质粒转染该细胞后,绿色荧光蛋白在细胞株中得到表达.反向插入报告基因的微复制子转染感染麻疹病毒的Vero/pcDNA3-T7细胞后,细胞中能够检测到报告基因的表达.用细胞系取代痘苗病毒系统,可以提高拯救效率.     

Hepatocyte-specific gene expression from integrated lentiviral vectors

BACKGROUND: For many applications, efficient gene therapy will require long-term, organ-specific therapeutic gene expression. Lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Many experimental vectors express transgenes under the control of the cytomegalovirus (CMV) immediate-early gene promoter. Although this promoter directs strong gene expression in vitro, it may be shut off rapidly in vivo. This study explores the potential of HIV-1-based vectors to transduce hepatocytes and compares gene expression from different promoters in integrated vectors. METHODS: HIV-1-based vector plasmids expressing the green fluorescent protein (GFP) under the control of the CMV promoter, the alpha-1 antitrypsin gene promoter or promoters derived from the hepatitis B virus (HBV) genome were used to compare expression in transfected and transduced cell lines. RESULTS: Hepatocyte cell lines differed strikingly in their transfectability. Transduction with replication-deficient HIV-1-based vector particles incorporating the different promoter elements was uniformly effective in hepatocyte and non-hepatocyte lines. However, in hepatocytes, only the CMV, alpha-1 antitrypsin and HBV core but not HBV surface promoters were able to produce GFP expression. Addition of the HBV enhancer 2 element improved the transducing ability of the HBV surface promoter and suppressed expression in non-hepatocytes increasing specificity for hepatocytes. CONCLUSIONS: Integrated lentiviral vectors can be used to direct transgene expression in liver cells both promiscuously and specifically. Promoters derived from the alpha-1 antitrypsin gene or HBV are alternatives to the CMV promoter. Inclusion of the HBV enhancer 2 permits strong liver-specific gene expression in vitro.     

Hybrid Cytomegalovirus-U6 Promoter-based Plasmid Vectors Improve Efficiency of RNA Interference in Zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.     

Enhancing expression level and stability of transgene mediated by episomal vector via buffering DNA methyltransferase in transfected CHO cells

Nonviral episomal vectors present attractive alternative vehicles for gene therapy applications. Previously, we have established a new type of nonviral episomal vector-mediated by the characteristic motifs of matrix attachment regions (MARs), which is driven by the cytomegalovirus (CMV) promoter. However, the CMV promoter is intrinsically susceptible to silencing, resulting in declined productivity during long-term culture. In this study, Chinese hamster ovary (CHO) cells and DNA methyltransferase-deficient (Dnmt3a-deficient) CHO cells were transfected with plasmid-mediated by MAR, or CHO cells were treated with the DNA methylation inhibitor 5-Aza-2′-deoxycytidine. Flow cytometry, plasmid rescue experiments, fluorescence in-situ hybridization (FISH), and bisulfite sequencing were performed to observe transgene expression, its state of existence, and the CpG methylation level of the CMV promoter. The results indicated that all DNA methylation inhibitor and methyltransferase deficient cells could increase transgene expression levels and stability in the presence or absence of selection pressure after a 60-generation culture. Plasmid rescue assay and FISH analysis showed that the vector still existed episomally after long-time culture. Moreover, a relatively lower CMV promoter methylation level was observed in Dnmt3a-deficient cell lines and CHO cells treated with 5-Aza-2′-deoxycytidine. In addition, Dnmt3a-deficient cells were superior to the DNA methylation inhibitor treatment regarding the transgene expression and long-term stability. Our study provides the first evidence that lower DNA methyltransferase can enhance expression level and stability of transgenes mediated by episomal vectors in transfected CHO cells.     

人SCF基因5′旁侧-1190～- 853 AT富集区功能研究

 已有的报告基因和EMSA实验研究表明 ,人干细胞生长因子 (SCF)基因 5′旁侧AT富集区- 1 1 90～ - 853在HeLa和MCF 7细胞中均能增强下游基因转录 ,可能为一个核基质结合区(MAR) ,对人SCF基因的转录发挥调控作用 .为进一步研究该AT富集区的功能 ,将人SCF基因 5′旁侧 - 1 1 90～ - 853AT富集区分别克隆入SV40或CMV启动子前后紧接着CAT报告基因 ,瞬时转染Jurkat,HepG2和 3T3细胞 ,检测CAT报告基因的瞬时表达活性 .结果表明 :人SCF基因 5′旁侧- 1 1 90～ - 853AT富集区在Jurkat和HepG2细胞中 ,对分别由SV40和CMV启动子引导的CAT基因表达均有抑制作用 ;但在 3T3细胞中对SV40启动子的转录活性表现出增强作用 ,对CMV启动子的转录活性无明显影响 .这些结果提示 ,人SCF基因 5′旁侧 - 1 1 90～ - 853AT富集区转录调控具有组织细胞特异性 ,在不同的细胞中可能发挥转录增强或抑制作用