【摘要】：羊癢病是一種傳染性海綿狀腦病(TSEs),是由正常的宿主細(xì)胞型朊蛋白構(gòu)象改變且在中樞神經(jīng)系統(tǒng)中積累引起的。山羊癢病是由以α螺旋為主的細(xì)胞型朊蛋白Pr Pc轉(zhuǎn)變?yōu)楦缓抡郫B的致病型朊蛋白Pr Psc引起的。Pr Pc基因(PRNP)的表達(dá)水平是癢病起始、發(fā)展和傳染的必要條件。關(guān)于山羊Pr Pc表達(dá)的轉(zhuǎn)錄調(diào)控研究尚不夠深入。本實驗利用生物信息學(xué)和比較基因組學(xué)對山羊PRNP基因的非翻譯區(qū)調(diào)控序列進(jìn)行分析;并結(jié)合熒光素酶報告基因系統(tǒng)探究山羊PRNP基因的啟動子區(qū)域。結(jié)果將為防控山羊癢病的發(fā)生提供重要的理論依據(jù)。本實驗中,對山羊PRNP基因(Gen Bank登陸號為EF870890.1)5’側(cè)翼序列和內(nèi)含子1序列特征進(jìn)行分析,并對這兩個區(qū)域進(jìn)行克隆與鑒定。利用分子克隆和亞克隆策略,構(gòu)建了山羊PRNP基因包含5’側(cè)翼區(qū)和外顯子1區(qū)域(3776-6107bp)的7個不同長度的缺失重組載體,同時構(gòu)建了包含內(nèi)含子1區(qū)域(5129-6891bp)的5個不同長度的缺失重組載體,利用雙熒光素酶報告系統(tǒng)在SH-SY5Y細(xì)胞中檢測其活性。結(jié)果表明山羊PRNP基因核心啟動子區(qū)域定位在4570-5171bp(包含外顯子1)。對該區(qū)域進(jìn)行轉(zhuǎn)錄因子結(jié)合位點預(yù)測,發(fā)現(xiàn)該區(qū)域有多個潛在轉(zhuǎn)錄因子結(jié)合位點,為山羊PRNP基因的關(guān)鍵啟動子區(qū)。在內(nèi)含子1區(qū)未發(fā)現(xiàn)具有啟動子活性的區(qū)域。對山羊PRNP基因核心啟動子區(qū)(4570-5171bp)進(jìn)一步進(jìn)行缺失,構(gòu)建了4個p GL3-缺失片段重組載體,利用雙熒光素酶報告系統(tǒng)在SH-SY5Y細(xì)胞中檢測其活性。結(jié)果表明核心啟動子區(qū)存在的4個motif不影響基因啟動子的活性。對內(nèi)含子1進(jìn)行缺失突變,表明在6002-7220bp內(nèi)存在抑制基因轉(zhuǎn)錄的轉(zhuǎn)錄因子結(jié)合位點。
[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 偽 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.
【學(xué)位授予單位】：河北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S827

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本文編號：2476697