人类心脏发育候选基因TRIM45及BZW2的蛋白相互作用研究及初步功能鉴定

发布时间：2018-01-09 14:04

本文关键词：人类心脏发育候选基因TRIM45及BZW2的蛋白相互作用研究及初步功能鉴定　出处：《湖南师范大学》2011年硕士论文　论文类型：学位论文

【摘要】：心脏的发育是一个极其复杂并且精细的过程。其主要调控网络由心源性转录因子和生长分化因子构成,这些基因的突变或异常表达会导致早期胚胎死亡或者各种心脏疾病。其中,心肌肥厚是比较常见的一种心脏病。其发病机制一直是心脏疾病机理研究领域的热点。 TRIM家族基因在多种细胞功能方面起着重要的作用,包括细胞增殖,分化,发展,发生以及细胞凋亡。本研究室前期克隆了人类TRIM45基因.基因全长3584bp。Northern B lot分析结果显示,TRIM45基因在成人的骨骼肌、脑、胰腺、心脏组织中依次有不同强度的表达。酵母双杂交结果显示TRIM45蛋白能够与SLC25A3蛋白以及DNAJB6蛋白发生相互作用。本文利用免疫共沉淀技术证明了TRIM45蛋白在体内能够与SLC25A3蛋白以及DNAJB6蛋白发生相互作用。DNAJB6蛋白属于热休克蛋白家族(Hsp家族)。已有文献表明,DNAJB6能够通过与NFATc3相互作用来阻断钙调磷酸酶诱导的心肌肥厚,并且DNAJB6能够抑制NFAT-Luc的荧光活性。本研究重复了DNAJB6对NFAT的转录活性调控研究,得到了与文献一致的结果。为了更进一步的了解TRIM45与心肌肥厚的关系,本研究利用荧光报告系统分别研究了TRIM45对心脏标志基因的转录活性调控。所用荧光报告质粒包括NFAT-Luc、SRF-Luc和MEF2C-Luc。结果显示,TRIM45能够强烈抑制ANF-Lu、NFAT-Luc、SRF-Luc和MEF2C-Luc的荧光活性。另外,SLC25A3也能强烈抑制NFAT-Luc的荧光活性。这些结果一起表明TRIM45, SLC25A3, DNAJB6都是心肌肥厚抑制因子,并且可能通过形成复合物来共同抑制心肌肥大。人类BZW2基因是一个新基因,其功能未知。它由bzip结构域和eIF5C结构域构成。BZW2基因在人类,小鼠,大鼠,斑马鱼,猩猩,狗,鸡等物种中均存在同源基因。BZW2基因定位于人类染色体7p21.1,在小鼠中定位于12；12B2。人类BZW2蛋白与小鼠BZW2蛋白同源度非常高,达到了99%。人类BZW2基因全长1883bp,编码419个氨基酸。本文用PCR技术扩增了人类BZW2基因的全长,将目的片段插入到原核表达载体pGEX-4T-1中,经过酶切和测序鉴定后,将重组质粒转入BL21感受态细胞,通过IPTG诱导表达融合蛋白,纯化后免疫新西兰大白兔。BZW2多克隆抗体效价为1：50。利用小鼠组织进行Western blot检测分析,结果显示BZW2在小鼠心脏,肌肉以及脑组织中高表达。通过生物信息学分析发现,BZW2蛋白能够与BZW1、PSTPIP1蛋白发生物理相互作用。通过荧光报告系统分析发现,BZW2能够抑制NF-kB和NFAT的转录活性,提示BZW2可能是一个潜在的心脏发育候选基因。
[Abstract]:Cardiac development is an extremely complex and delicate process. Its main regulatory network is composed of cardiogenic transcription factors and growth and differentiation factors. Mutations or abnormal expression of these genes can lead to early embryonic death or various heart diseases. Myocardial hypertrophy is a common heart disease, and its pathogenesis has been a hot spot in the field of heart disease. TRIM family genes play an important role in many cell functions, including cell proliferation, differentiation and development. The human TRIM45 gene was cloned in the early stage of this study. The full-length of the gene was 3584 bp.Northern B lot analysis. The TRIM45 gene is present in adult skeletal muscle, brain, and pancreas. The results of yeast two-hybrid showed that TRIM45 protein could interact with SLC25A3 protein and DNAJB6 protein. The results show that TRIM45 protein can interact with SLC25A3 protein and DNAJB6 protein in vivo. DNA JB6 protein belongs to heat shock protein family. Hsp family. DNAJB6 can block the cardiac hypertrophy induced by calmodulase by interacting with NFATc3. Moreover, DNAJB6 can inhibit the fluorescence activity of NFAT-Luc. In this study, the regulation of NFAT transcriptional activity by DNAJB6 was repeated. In order to further understand the relationship between TRIM45 and myocardial hypertrophy. In this study, we studied the transcriptional activity of TRIM45 on cardiac marker genes by fluorescence reporting system. The fluorescent reporter plasmids including NFAT-Luc were used. The results of SRF-Luc and MEF2C-Luc. showed that TRIM45 could strongly inhibit ANF-Lun NFAT-Luc. The fluorescence activity of SRF-Luc and MEF2C-Luc. In addition, SLC25A3 also strongly inhibited the fluorescence activity of NFAT-Luc. These results indicated that TRIM45. SLC25A3, DNAJB6 are myocardial hypertrophy suppressor, and may inhibit myocardial hypertrophy by forming complex. Human BZW2 gene is a new gene whose function is unknown. It consists of bzip domain and eIF5C domain. BZW2 gene is composed of human, mouse, rat, zebrafish, gorilla, dog. The homologous gene. BZW2 was located in human chromosome 7p21. 1 and in mice. 12B2.The homology between human BZW2 protein and mouse BZW2 protein is very high, reaching 99%. The length of human BZW2 gene is 1883 BP. The full length of human BZW2 gene was amplified by PCR. The target fragment was inserted into prokaryotic expression vector pGEX-4T-1 and identified by restriction endonuclease digestion and sequencing. The recombinant plasmid was transferred into BL21 receptive cells and the fusion protein was induced by IPTG. The polyclonal antibody titer of purified New Zealand white rabbit. BZW2 polyclonal antibody was 1: 50. Western blot analysis was carried out in mouse tissues. The results showed that BZW2 was in the heart of mice. It was found that BZW2 protein could interact with BZW1PSTPIP1 protein by bioinformatics analysis, and it was found by fluorescence report system analysis that BZW1PSTPIP1 protein could interact with BZW1PSTPIP1 protein. BZW2 can inhibit the transcriptional activity of NF-kB and NFAT, suggesting that BZW2 may be a potential candidate gene for cardiac development.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R394

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