山羊朊蛋白基因（PRNP）调控区转录因子结合位点的筛选
发布时间：2019-05-14 12:02
【摘要】：羊痒病是一种传染性海绵状脑病(TSEs),是由正常的宿主细胞型朊蛋白构象改变且在中枢神经系统中积累引起的。山羊痒病是由以α螺旋为主的细胞型朊蛋白Pr Pc转变为富含β折叠的致病型朊蛋白Pr Psc引起的。Pr Pc基因(PRNP)的表达水平是痒病起始、发展和传染的必要条件。关于山羊Pr Pc表达的转录调控研究尚不够深入。本实验利用生物信息学和比较基因组学对山羊PRNP基因的非翻译区调控序列进行分析;并结合荧光素酶报告基因系统探究山羊PRNP基因的启动子区域。结果将为防控山羊痒病的发生提供重要的理论依据。本实验中,对山羊PRNP基因(Gen Bank登陆号为EF870890.1)5’侧翼序列和内含子1序列特征进行分析,并对这两个区域进行克隆与鉴定。利用分子克隆和亚克隆策略,构建了山羊PRNP基因包含5’侧翼区和外显子1区域(3776-6107bp)的7个不同长度的缺失重组载体,同时构建了包含内含子1区域(5129-6891bp)的5个不同长度的缺失重组载体,利用双荧光素酶报告系统在SH-SY5Y细胞中检测其活性。结果表明山羊PRNP基因核心启动子区域定位在4570-5171bp(包含外显子1)。对该区域进行转录因子结合位点预测,发现该区域有多个潜在转录因子结合位点,为山羊PRNP基因的关键启动子区。在内含子1区未发现具有启动子活性的区域。对山羊PRNP基因核心启动子区(4570-5171bp)进一步进行缺失,构建了4个p GL3-缺失片段重组载体,利用双荧光素酶报告系统在SH-SY5Y细胞中检测其活性。结果表明核心启动子区存在的4个motif不影响基因启动子的活性。对内含子1进行缺失突变,表明在6002-7220bp内存在抑制基因转录的转录因子结合位点。
[Abstract]:Sheep itching is an infectious spongiform encephalopathy (TSEs), is caused by the conformational change of normal host cell prion protein and its accumulation in the central nervous system. Goat itching is a necessary condition for the initiation, development and transmission of goat itching, which is caused by the transformation of Pr Pc, a cellular prion protein dominated by 伪 helix, to the expression of. Pr Pc gene (PRNP), which is rich in 尾-fold pathogenic prion protein Pr Psc. The transcriptional regulation of goat Pr Pc expression is not deep enough. In this experiment, the untranslated region regulatory sequences of goat PRNP gene were analyzed by bioinformatics and comparative genomics, and the promoter region of goat PRNP gene was investigated by luciferase reporter gene system. The results will provide an important theoretical basis for the prevention and control of goat itching. In this experiment, the 5 'flanking sequence and intron 1 sequence of goat PRNP gene (Gen Bank landing number EF870890.1 were analyzed, and the two regions were cloned and identified. Seven deletion recombinant vectors containing 5 'flanking region and exon 1 region (3776-6107bp) of goat PRNP gene were constructed by molecular cloning and subcloning strategy. At the same time, five deletion recombinant vectors containing intron 1 region (5129-6891bp) were constructed and their activity was detected in SH-SY5Y cells by double luciferase reporting system. The results showed that the core promoter region of goat PRNP gene was located in 4570-5171bp (including Exon 1). The transcriptional factor binding sites in this region were predicted. It was found that there were several potential transcription factor binding sites in this region, which was the key promoter region of goat PRNP gene. No promoter activity region was found in intron 1 region. The core promoter region (4570-5171bp) of goat PRNP gene was further deleted. Four recombinant vectors of p GL3- deletion fragment were constructed and their activity was detected in SH-SY5Y cells by double luciferase reporting system. The results showed that the activity of gene promoter was not affected by the existence of four motif in the core promoter region. The deletion mutation of intron 1 indicates that there are transcription factor binding sites that inhibit gene transcription in 6002-7220bp.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827
本文编号：2476697
[Abstract]:Sheep itching is an infectious spongiform encephalopathy (TSEs), is caused by the conformational change of normal host cell prion protein and its accumulation in the central nervous system. Goat itching is a necessary condition for the initiation, development and transmission of goat itching, which is caused by the transformation of Pr Pc, a cellular prion protein dominated by 伪 helix, to the expression of. Pr Pc gene (PRNP), which is rich in 尾-fold pathogenic prion protein Pr Psc. The transcriptional regulation of goat Pr Pc expression is not deep enough. In this experiment, the untranslated region regulatory sequences of goat PRNP gene were analyzed by bioinformatics and comparative genomics, and the promoter region of goat PRNP gene was investigated by luciferase reporter gene system. The results will provide an important theoretical basis for the prevention and control of goat itching. In this experiment, the 5 'flanking sequence and intron 1 sequence of goat PRNP gene (Gen Bank landing number EF870890.1 were analyzed, and the two regions were cloned and identified. Seven deletion recombinant vectors containing 5 'flanking region and exon 1 region (3776-6107bp) of goat PRNP gene were constructed by molecular cloning and subcloning strategy. At the same time, five deletion recombinant vectors containing intron 1 region (5129-6891bp) were constructed and their activity was detected in SH-SY5Y cells by double luciferase reporting system. The results showed that the core promoter region of goat PRNP gene was located in 4570-5171bp (including Exon 1). The transcriptional factor binding sites in this region were predicted. It was found that there were several potential transcription factor binding sites in this region, which was the key promoter region of goat PRNP gene. No promoter activity region was found in intron 1 region. The core promoter region (4570-5171bp) of goat PRNP gene was further deleted. Four recombinant vectors of p GL3- deletion fragment were constructed and their activity was detected in SH-SY5Y cells by double luciferase reporting system. The results showed that the activity of gene promoter was not affected by the existence of four motif in the core promoter region. The deletion mutation of intron 1 indicates that there are transcription factor binding sites that inhibit gene transcription in 6002-7220bp.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827
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