BAT1基因?qū)嵝菘艘蜃親sf4b的作用
發(fā)布時間：2019-06-26 08:57
【摘要】：背景 白內(nèi)障,又稱眼球晶狀體的部分或全部渾濁化,可導(dǎo)致視力下降,大多數(shù)表現(xiàn)為視覺靈敏度、相對靈敏度下降,以及眩光�？煞譃樵绨l(fā)型(先天性)白內(nèi)障和遺傳性白內(nèi)障。先天性白內(nèi)障危害特別嚴(yán)重,因為其有可能抑制視力的發(fā)育,導(dǎo)致永久失明。 在過去的十年間,通過在大家族中使用遺傳連鎖分析策略,對先天性白內(nèi)障的遺傳背景的了解已有了相當(dāng)大的進(jìn)步。大約有40多個和白內(nèi)障相關(guān)的遺傳位點已被知曉,其中的25個已經(jīng)過鑒定,而突變位點的數(shù)量已超過100。突變導(dǎo)致的發(fā)育性白內(nèi)障主要涉及一些結(jié)構(gòu)和伴侶蛋白,包括α-,β-,γ-晶體蛋白。另外一組包括晶狀體特異性跨膜間隙連接蛋白GJA3和GJA8,以及膜蛋白MIP和LIM2.第三組包括一些和晶狀體相關(guān)的轉(zhuǎn)錄因子蛋白,如HSF4,PITX3,MAF, PAX6和FOXE3。 在人體內(nèi),hsf4基因的錯義突變可導(dǎo)致白內(nèi)障,而帶有靶向hsf4基因缺失的小鼠,表現(xiàn)出晶狀體纖維細(xì)胞分化上的缺陷和早期的白內(nèi)障癥狀。HSF4屬于熱休克轉(zhuǎn)錄因子家族(HSF),能和熱休克原件結(jié)合(HSE),在外界不同刺激的情況下轉(zhuǎn)錄激活下游的熱休克蛋白(HSP70,HSP90,HSP27,HSP82)。HSF4擁有兩個剪切變體：Hsf4a有轉(zhuǎn)錄抑制活性,而Hsf4b具有轉(zhuǎn)錄激活作用。近來的研究發(fā)現(xiàn)Hsf4b在晶狀體纖維細(xì)胞的成熟中起到重要作用。雖然Hsf4的作用已經(jīng)明了,但是其調(diào)控轉(zhuǎn)錄的機(jī)制還不是太清楚。 為了進(jìn)一步了解控制Hsf4b轉(zhuǎn)錄活性的分子機(jī)制,我們進(jìn)行了酵母雙雜交試驗,用Hsf4b作為誘餌,發(fā)現(xiàn)了一個新的和Hsf4b反應(yīng)的蛋白BAT1(又名BAT1,56kDaU2AF65-associated protein)。BAT1蛋白作為DEAD盒式家族的一員,是一個具有ATP依賴的RNA解旋酶。BAT1既具有RNA激活的ATP結(jié)合和水解活性,又具有ATP依賴的RNA解旋活性。研究不同組織中的BAT1蛋白發(fā)現(xiàn),BAT1蛋白不僅在前體mRNA的剪切中起到重要作用,而且在mRNA的核內(nèi)輸出及細(xì)胞質(zhì)mRNA的定位上也有重要作用。 本實驗通過構(gòu)建一系列表達(dá)BAT1蛋白的載體,來驗證-BAT1和hsf4b的相互作用。然后通過luciferase assay實驗來驗證-BAT1對hsf4b下游基因的調(diào)控作用。 目的 本研究通過構(gòu)建表達(dá)BAT1蛋白的一系列載體,來驗證-BAT1和hsf4b的相互作用,確定其作用部位,以及BAT1對hsf4b下游相關(guān)基因的調(diào)控作用。 方法 1.應(yīng)用PCR技術(shù),獲得表達(dá)BAT1的cDNA序列,利用Bamh I和Not I酶切位點將BAT1基因分別克隆入真核表達(dá)載體pEBG, pcDNA3.0,得到pEBG-BAT1, pcDNA3.0-T7-BAT1質(zhì)粒。利用Bamh Ⅰ和Sal I酶切位點將BAT1基因克隆入pGEX-4T3—4T3原核表達(dá)載體,得到pGEX-4T3-BAT1質(zhì)粒。 2.測序證確后,將上述pEBG-BAT1, pcDNA3.0-T7-BAT1質(zhì)粒轉(zhuǎn)染293T細(xì)胞,進(jìn)行真核表達(dá)。利用SDS-PAGE和Western blot實驗鑒定體內(nèi)BAT1蛋白的表達(dá)。將pGEX-4T3-BAT1質(zhì)粒轉(zhuǎn)入BL21感受態(tài)細(xì)胞,誘導(dǎo)表達(dá)BAT1蛋白的克隆。利用SDS-PAGE實驗鑒定體外表達(dá)的BAT1蛋白。 3.通過體內(nèi),體外pull-down assay來驗證BAT1蛋白和hsf4b的蛋白相互作用,以及通過pull-down assay來驗證BAT1和hsf4b的哪個功能區(qū)作用。 4.通過luciferase assay實驗,來驗證BAT1對hsf4b下游基因α B-晶體蛋白(alpha B-crystallin)的影響。 結(jié)果 1.構(gòu)建了表達(dá)BAT1的真核載體pEBG-BAT1, pcDNA3.0-T7-BAT1,原核載體pGEX-4T3-BAT1,并獲得了相應(yīng)的表達(dá)蛋白。 2.體內(nèi)pull-down assay實驗驗證了BAT1和hsf4b具有相互作用；體外pull-down assay實驗也驗證了BAT1和hsf4b具有相互作用。并且初步證明了BAT1和hsf4b的C-端功能區(qū)有相互作用。 3.Luciferase assay實驗初步說明了BAT1對hsf4b下游基因α B-晶體蛋白(alpha B-crystallin)有抑制作用。 結(jié)論 1.BAT1蛋白和hsf4b在體內(nèi)和體外都有結(jié)合作用 2.BAT1蛋白和hsf4b的C-端功能區(qū)有相互作用 3.BAT1對hsf4b下游基因α B-晶體蛋白(alpha B-crystallin)的表達(dá)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
【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R776.1
[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
【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R776.1
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