BAT1基因对热休克因子Hsf4b的作用
[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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