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BAT1基因对热休克因子Hsf4b的作用

发布时间:2019-06-26 08:57
【摘要】:背景 白内障,又称眼球晶状体的部分或全部浑浊化,可导致视力下降,大多数表现为视觉灵敏度、相对灵敏度下降,以及眩光。可分为早发型(先天性)白内障和遗传性白内障。先天性白内障危害特别严重,因为其有可能抑制视力的发育,导致永久失明。 在过去的十年间,通过在大家族中使用遗传连锁分析策略,对先天性白内障的遗传背景的了解已有了相当大的进步。大约有40多个和白内障相关的遗传位点已被知晓,其中的25个已经过鉴定,而突变位点的数量已超过100。突变导致的发育性白内障主要涉及一些结构和伴侣蛋白,包括α-,β-,γ-晶体蛋白。另外一组包括晶状体特异性跨膜间隙连接蛋白GJA3和GJA8,以及膜蛋白MIP和LIM2.第三组包括一些和晶状体相关的转录因子蛋白,如HSF4,PITX3,MAF, PAX6和FOXE3。 在人体内,hsf4基因的错义突变可导致白内障,而带有靶向hsf4基因缺失的小鼠,表现出晶状体纤维细胞分化上的缺陷和早期的白内障症状。HSF4属于热休克转录因子家族(HSF),能和热休克原件结合(HSE),在外界不同刺激的情况下转录激活下游的热休克蛋白(HSP70,HSP90,HSP27,HSP82)。HSF4拥有两个剪切变体:Hsf4a有转录抑制活性,而Hsf4b具有转录激活作用。近来的研究发现Hsf4b在晶状体纤维细胞的成熟中起到重要作用。虽然Hsf4的作用已经明了,但是其调控转录的机制还不是太清楚。 为了进一步了解控制Hsf4b转录活性的分子机制,我们进行了酵母双杂交试验,用Hsf4b作为诱饵,发现了一个新的和Hsf4b反应的蛋白BAT1(又名BAT1,56kDaU2AF65-associated protein)。BAT1蛋白作为DEAD盒式家族的一员,是一个具有ATP依赖的RNA解旋酶。BAT1既具有RNA激活的ATP结合和水解活性,又具有ATP依赖的RNA解旋活性。研究不同组织中的BAT1蛋白发现,BAT1蛋白不仅在前体mRNA的剪切中起到重要作用,而且在mRNA的核内输出及细胞质mRNA的定位上也有重要作用。 本实验通过构建一系列表达BAT1蛋白的载体,来验证-BAT1和hsf4b的相互作用。然后通过luciferase assay实验来验证-BAT1对hsf4b下游基因的调控作用。 目的 本研究通过构建表达BAT1蛋白的一系列载体,来验证-BAT1和hsf4b的相互作用,确定其作用部位,以及BAT1对hsf4b下游相关基因的调控作用。 方法 1.应用PCR技术,获得表达BAT1的cDNA序列,利用Bamh I和Not I酶切位点将BAT1基因分别克隆入真核表达载体pEBG, pcDNA3.0,得到pEBG-BAT1, pcDNA3.0-T7-BAT1质粒。利用Bamh Ⅰ和Sal I酶切位点将BAT1基因克隆入pGEX-4T3—4T3原核表达载体,得到pGEX-4T3-BAT1质粒。 2.测序证确后,将上述pEBG-BAT1, pcDNA3.0-T7-BAT1质粒转染293T细胞,进行真核表达。利用SDS-PAGE和Western blot实验鉴定体内BAT1蛋白的表达。将pGEX-4T3-BAT1质粒转入BL21感受态细胞,诱导表达BAT1蛋白的克隆。利用SDS-PAGE实验鉴定体外表达的BAT1蛋白。 3.通过体内,体外pull-down assay来验证BAT1蛋白和hsf4b的蛋白相互作用,以及通过pull-down assay来验证BAT1和hsf4b的哪个功能区作用。 4.通过luciferase assay实验,来验证BAT1对hsf4b下游基因α B-晶体蛋白(alpha B-crystallin)的影响。 结果 1.构建了表达BAT1的真核载体pEBG-BAT1, pcDNA3.0-T7-BAT1,原核载体pGEX-4T3-BAT1,并获得了相应的表达蛋白。 2.体内pull-down assay实验验证了BAT1和hsf4b具有相互作用;体外pull-down assay实验也验证了BAT1和hsf4b具有相互作用。并且初步证明了BAT1和hsf4b的C-端功能区有相互作用。 3.Luciferase assay实验初步说明了BAT1对hsf4b下游基因α B-晶体蛋白(alpha B-crystallin)有抑制作用。 结论 1.BAT1蛋白和hsf4b在体内和体外都有结合作用 2.BAT1蛋白和hsf4b的C-端功能区有相互作用 3.BAT1对hsf4b下游基因α B-晶体蛋白(alpha B-crystallin)的表达有抑制作用
[Abstract]:background Cataract, also known as the partial or all of the opacification of the lens of the eye, can result in a decrease in vision, most of which are visual acuity, reduced relative sensitivity, and dizziness Light. It can be divided into early-style (congenital) cataracts and hereditary white The congenital cataract is particularly severe, as it may inhibit the development of vision, leading to permanent loss The understanding of the genetic background of congenital cataract has been considerable over the last 10 years by using a genetic linkage analysis strategy in the large family. Progress has been made. Some 40 and cataract-related genetic sites have been known,25 of which have been identified, and the number of mutation sites has exceeded 100. The developmental cataracts resulting from the mutation mainly involve some of the structural and chaperone proteins, including the 1-,1-,1-, Body proteins. The other group includes lens-specific transmembrane clearance-binding proteins GJA3 and GJA8, and membrane proteins MIP and L. IM2. The third group includes some and lens-related transcription factor proteins, such as HSF4, PITX3, MAF, PAX6 and FO XE3. In the human body, the missense mutation of the hff4 gene can lead to the cataract, and the mouse with the target hsc4 gene is deleted, and the defect and the early stage of the differentiation of the lens are shown. The HSF4 belongs to the heat shock transcription factor family (HSF), can be combined with the heat shock original (HSE), and the heat shock protein (HSP70, HSP90, HSP27, H) downstream can be transcribed and activated under different external stimuli. SP82). HSF4 has two shear variants: Hsf4a has transcriptional inhibitory activity and Hsf4b has a rotational speed recording and activation. Recent studies have found that Hsf4b plays a role in the maturation of the lens It is important to play an important role. Although the effect of Hsf4 is clear, the mechanism for regulating and controlling transcription further It is not too clear. In order to further understand the molecular mechanism for controlling the transcription activity of Hsf4b, we carried out a yeast two-hybridization assay, using Hsf4b as a bait, a new protein BAT1 (also known as BAT1,56 kDaU2AF65-associated p) that reacted with Hsf4b was found. rotein). The BAT1 protein is a member of the DEAD cassette family and is an ATP dependent The RNA helicase. BAT1 has both the ATP binding and the hydrolytic activity of the RNA activation, but also the ATP-dependent It is found that the BAT1 protein not only plays an important role in the cleavage of the precursor mRNA, but also the localization of the cytoplasmic mRNA in the nucleus of the mRNA. It is also important to construct a series of vectors expressing the BAT1 protein to verify the-BAT1 and h The interaction of sf4b. Then verify the-BAT1 pair hsf4b by the lucifer assay test. downstream The purpose of this study was to verify the interaction between-BAT1 and hsf4b by constructing a series of vectors expressing the BAT1 protein, to determine the action site, and to BAT1 to hsf. 4b and 1, using the PCR technology to obtain a cDNA sequence for expressing BAT1, and cloning the BAT1 gene into the eukaryotic expression vector pEBG and the pcDNA3 by using the Bamh I and the Not I restriction site to obtain pEBG-BAT1, p, The BAT1 gene was cloned into the prokaryotic expression vector of pGEX-4T3-4T3 by using BamH I and Sal I restriction sites. The pGEX-4T3-BAT1 plasmid was obtained.2. After the sequencing, the above pEBG-BAT1, pcDNA3.0-T7-BA A T1 plasmid was transfected into 293T cells for eukaryotic expression. SDS-PAGE and Western blotting were used. The expression of the BAT1 protein in the body was identified by Western blot. The pGEX-4T3-BAT1 plasmid was transferred to the BL21. Competent cells to induce the expression of a clone of the BAT1 protein. 3. The protein interaction between the BAT1 protein and the hsf4b was verified by the in vitro and in vitro pull-down assay, and the expression of the BAT1 protein was verified by a full-down assay. To verify which functional area of the BAT1 and hsf4b.4.4. Verify that the BAT1 is a B-crystal egg downstream of the hsf4b by the lucifer assay. white ( Results 1. The eukaryotic expression vector pEBG-BAT1, pcDNA3.0-T7-BAT1 and the prokaryotic expression of BAT1 were constructed. The expression of pGEX-4T3-BAT1 and the corresponding expression of pGEX-4T3-BAT1 were obtained. The wnassay experiment also verified that BAT1 and hsf4b had an interaction. And the C-terminal functional area of BAT1 and hsf4b is preliminarily proved to have an interaction.3. Lucifasay assay is a preliminary illustration of the downstream gene of BAT1 to hsf4b. A. B. -Crystal protein (alpha B-crystanin) Conclusion 1. BAT1 protein and hsf4b in vivo in vitro and in vitro there is a binding action of 2. The C-terminal functional area of the BAT1 protein and hsf4b has an interaction 3. BAT1 vs. hsf
【学位授予单位】:河南大学
【学位级别】:硕士
【学位授予年份】:2012
【分类号】:R776.1

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