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,24h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。     

miR-122过表达转基因小鼠质粒构建及其功能验证

目的:比较两种miR-122转基因小鼠过表达载体构建方法,为建立miR-122过表达转基因小鼠奠定基础。方法:PCR扩增长约291bp的pre-miR-122的序列,分别定向克隆到pBROAD3-GFP载体GFP基因上游内含子或下游3'UTR区域,两种质粒分别转染293T细胞,Q-PCR检测miR-122和GFP的表达水平,并观察GFP绿色荧光。miR-122 sensor reporter是将3个miR-122成熟序列的反义序列串联克隆至psiCHECK2载体luciferase 3'UTR中,然后分别与2种miR-122过表达质粒载体共转染293T细胞,最后检测荧光素酶活性来鉴定miR-122调控功能。结果:2种构建方法的miR-122表达水平都明显增高,而只有插入到GFP基因3'UTR的质粒表达GFP功能正常。结论:构建microRNA过表达载体时,microRNA位于报告基因3'UTR区域不会影响microRNA和报告基因的功能;构建的两种miR-122过表达质粒载体都可应用到转基因小鼠研究中,而将miR-122插入到GFP下游的方法则更利于miR-122的表达。     

β-萘黄酮能抑制荧光素酶活性

目的：研究β-萘黄酮对荧光索酶活性的影响。方法：利用人GCLC基因调控序列驱动的GCLC．PGL3．enhancer-Luciferase报道载体（PL45）转染人肺腺癌细胞A549,人肝癌细胞HepG2,人子宫颈癌细胞HeLa,人乳腺癌细胞MCF-7,人肝癌细胞Bel-7402,人支气管上皮细胞16HBE,β-萘黄酮刺激后,双荧光素酶报告基因检测系统分析其对GCLC基因表达的影响。westerblot检测β-NF刺激16HBE细胞后GCLC蛋白水平的变化。β-萘黄酮刺激转染了表达Luciferase的真核表达载体pRC／CMV2．1uc＋的A549和HepG2细胞后,双荧光素酶报告基因检测系统分析其对Luciferase基因表达的影响。PIA5转染A549和HepG2细胞,裂解细胞后用p-NF刺激,双荧光素酶报告基因检测系统分析其对Luciferase基因的影响。结果：在各种细胞中,转染PL45报道载体后,β-NF处理组荧光素酶相对活性值与DMSO对照组相比均明显下降（p〈0．01）。westerblot结果显示β-NF处理组GCLC蛋白的表达较DMsO对照蛆明显升高。在A549和HepG2细胞中,转染pRC／CMV2．1uc＋载体后,β-NF处理组荧光素酶相对活性值与DMSO对照组相比均明显下降（P〈0．01）。PIA5转染A549和HepG2细胞,裂解细胞后用β-NF刺激,β-NF处理组荧光素酶相对活性值与DMSO对照组相比均明显下降（P〈0．01）。结论：β-萘黄酮直接抑制了荧光素酶的活性     

Secretion of firefly luciferase in E.coli

Summary A DNA segment carrying the full-length, intronless firefly luciferase gene was inserted into the high expression secretion vector, pIN-III -ompA. Upon induction of gene expression, luciferase activity was detected in extracts prepared from periplasmic fractions. The results indicated that the OmpA signal peptide was able to direct secretion of firefly luciferase across the cytoplasmic membrane. This has important implications for using this luciferase as a reporter in studying protein export and targeting.     

HCVIRES介导萤火虫荧光素酶翻译的双顺反子载体的构建与在小鼠体内的表达

将HCVIRES插入双报告基因海肾荧光素酶 (Rluc)基因和萤火虫荧光素酶 (Fluc)基因之间 ,建立了“依赖帽子的扫描机制”翻译表达Rluc ,HCVIRES调控Fluc翻译的双顺反子表达载体pCI Rluc HCVIRES Fluc ,通过酶切反应及转染HepG2细胞鉴定双荧光素酶瞬间表达活性等试验 ,证实获得了表达双荧光素酶的双顺反子载体 .并应用水压转染法将双顺反子表达质粒导入小鼠体内 ,在小鼠肝脏检测到高水平表达的Rluc和Fluc .该研究成功构建一种HCVIRES介导萤火虫荧光素酶基因表达的双顺反子载体 ,并在HepG2细胞及小鼠体内进行了瞬时表达 ,为进一步建立稳定评价靶向HCVIRES药物作用的细胞及小动物模型研究奠定了基础     

Amastin mRNA abundance in Trypanosoma cruzi is controlled by a 3'-untranslated region position-dependent cis-element and an untranslated region-binding protein

The genome of Trypanosoma cruzi contains tandem arrays of alternating genes encoding amastin and tuzin. Amastin is a surface glycoprotein abundantly expressed on the intracellular mammalian amastigote form of the protozoan parasite, and tuzin is a G-like protein. We demonstrated previously that the amastin-tuzin gene cluster is polycistronically transcribed to an equal extent in all parasite life cycle stages. The steady state level of amastin mRNA, however, is 68-fold more abundant in amastigotes than in epimastigotes. Here we show that the half-life of amastin mRNA is 7 times longer in amastigotes than in epimastigotes. Linker replacement experiments demonstrate that the middle one-third of the 630-nucleotide 3'-untranslated region (UTR) is responsible for the amastin mRNA up-regulation. This positive effect is dependent on the distance of the 3'-UTR segment from the stop codon and the polyadenylation site as well as on its orientation. A protein or protein complex more abundant in amastigotes than in epimastigotes binds to this minimally defined 3'-UTR segment and may be involved in its regulatory function.     

热休克转录因子1对高迁移率族蛋白B1的转录调控作用及机制

目的：探讨烧伤血清诱导下热休克转录因子1（HSF1）对高迁移率族蛋白B1（HMGB1）的转录调控作用及其机制.方法：构建热休克转录因子1真核表达载体pcDNA3.1-HSF1,HMGB1野生型启动子荧光素酶报告基因pGL3-HMGB1-Y,HMGB1突变型启动子荧光素酶报告基因pGL3-HMGB1-T.pcDNA3.1-HSF1转染巨噬细胞RAW264.7,烧伤血清诱导后,半定量RT-PCR检测HMGB1 mRNA的表达.pcDNA3.1-HSF1,HMGB1启动子荧光素酶报告基因共转染RAW264.7,烧伤血清诱导后,检测并比较pGL3-HMGB1-Y和 pGL3-HMGB1-T的相对萤光素酶活性.结果：烧伤血清诱导下,HSF1可以下调RAW264.7 HMGB1mRNA的表达.pGL3-HMGB1-T的相对荧光素酶值较pGL3-HMGB1-Y明显下降,P＜0.01,差异有显著统计学意义.结论：烧伤血清诱导下,HSF1可能通过与HMGB1启动子区HSE的结合下调小鼠巨噬细胞RAW264.7 HMGB1mRNA的表达.     

CKLFSF1基因与CKLFSF2基因间存在的顺式作用元件

探讨趋化素样因子超家族成员 1,2基因 (CKLFSF1基因与CKLFSF2基因 )间的短序列对其下游基因表达的调控作用 .运用PCR技术扩增CKLFSF1基因与CKLFSF2基因间的序列 ,将此片段插入含有萤光素酶 (luciferase)报告基因载体上 .以磷酸钙介导基因转染技术 ,将重组质粒以及阴性和阳性对照组质粒转染到HeLa细胞 ,进行瞬时表达分析 .在pGL3 Basic质粒中的报告基因萤光素酶无表达 ,但将CKLFSF1与CKLFSF2基因间的序列插入到启动子上游或下游后 ,显著抑制其下游基因的表达 ,萤光素酶活性明显降低 .结果提示 ,CKLFSF1与CKLFSF2基因间的序列不具有启动子活性 ,但是该序列对其下游基因表达具有负调控作用     

Connexin43 mRNA contains a functional internal ribosome entry site

A reporter gene construct was used to study the regulation of connexin43 (Cx43) expression, the major gap junction protein found in heart and uterus, in transfected cell lines. The construct had the firefly luciferase gene under the control of the Cx43 promoter. Inclusion of the 5'-untranslated region (UTR) of the mRNA in the construct increased luciferase expression by 70%. A bicistronic vector assay demonstrated that the Cx43 5'-UTR contains a strong internal ribosome entry site (IRES). Deletion analysis localized the IRES element to the upstream portion of the 5'-UTR.