P53下游候选基因PAP1的鉴定和生物信息学分析

发布时间：2018-03-20 20:55

  本文选题：p53　切入点：下游基因　出处：《重庆大学》2006年硕士论文　论文类型：学位论文

【摘要】： p53基因是与人类肿瘤相关性最高的抑癌基因。前人研究表明, p53基因行使其生物学效应主要是通过P53蛋白与特异基因组序列结合,激活下游基因转录、作用来实现的。因此,鉴定p53下游基因是了解p53基因调控网络的关键。 本实验室在前期工作中建立携带野生型p53转基因的U251-pTet-p53细胞系,在四环素应答元件的控制下,诱导p53基因表达。通过差异显示技术,克隆到一个新的P53下游基因候选基因——PAP1基因。 本文构建了pET28b-p53重组表达质粒,将pET28b-p53转化到大肠杆菌BL21(DE3),IPTG诱导表达P53蛋白。用地高辛标记PAP1基因作为探针,与P53蛋白做凝胶滞留实验,鉴定P53蛋白下游候选基因PAP1。然后综合运用生物信息学方法对PAP1基因进行了基因结构分析和功能分析。 研究结果显示:(1)该基因序列能与野生型P53蛋白结合,与突变型P53蛋白几乎不结合。因此PAP1基因是P53蛋白的下游基因,受P53蛋白调控。(2)PAP1基因定位于人类染色体16p12-13上,PAP1蛋白分子量为32.9KD,理论等电点pI为5.81,化学方程式为C1505H2309N385O421S11,半衰期为30小时左右,不稳定指数:42.58,脂肪族指数Aliphatic index:95.64,总平均疏水性:-0.162。PAP1蛋白为亲水性蛋白,存在一个跨膜区,大约在42-79氨基酸片段,没有信号肽。(3)PAP1基因属于DREV1转甲基酶基因家族。PAP1mRNA对GenBank数据库进行相似性搜索-BLASTN,与DREV1基因的相似性很高,将它们进一步做双序列比对发现它们的相似性在80.8%,除了在开头和结尾的相似性很差之外,其它部分相似性较高。PAP1与DREV1,CD4,TCRα、β、γ多序列比对时,存在几个保守的位点。PAP1与各个物种的同源基因的多序列比对,它们的保守性很高。(4)PAP1蛋白的二级结构为混合型,40%为α螺旋,17%为β折叠,43%为其它类型的二级结构;含有DREV1转甲基酶结构域,同时PAP1蛋白也存在N-糖基化位点,蛋白酶C磷酸化位点,干酪素蛋白激酶Ⅱ磷酸化位点,N-十四酰基化位点,酪氨算盐酸化作用位点。(5)与老鼠IGSF6基因同源性较高,可能是同源基因。IGSF6基因在老鼠胚胎发育中起着重要作用,依此推测PAP1基因也可能在人的胚胎发育中起着重要的作用。
[Abstract]:P53 gene is the most associated tumor suppressor gene in human tumor. Previous studies have shown that p53 gene exerts its biological effect mainly through p53 protein binding with specific genomic sequence, activating downstream gene transcription and action. Identification of p53 downstream gene is the key to understand p53 gene regulatory network. The U251-pTet-p53 cell line carrying wild-type p53 gene transgene was established in our laboratory to induce p53 gene expression under the control of tetracycline response element. A novel candidate gene for P 53 downstream gene, PAP1 gene, was cloned. In this paper, pET28b-p53 recombinant expression plasmid was constructed and transformed into E. coli BL21DE3PTG to induce the expression of p53 protein. Digoxigenin-labeled PAP1 gene was used as a probe to conduct gel retention test with p53 protein. The downstream candidate gene PAP1 of p53 protein was identified. Then the gene structure and function of PAP1 gene were analyzed by bioinformatics. The results showed that the gene sequence could bind to wild-type p53 protein and hardly to mutant p53 protein. Therefore, PAP1 gene is the downstream gene of p53 protein. The molecular weight of PAP1 protein is 32.9KD, the theoretical isoelectric point Pi is 5.81, the chemical equation is C1505H2309N385O421S11, and the half-life is about 30 hours, and the molecular weight of PAP1 protein is 32.9KDand the theoretical isoelectric point Pi is 5.81.The molecular weight of PAP1 protein is 32.9 KD.The chemical equation is C1505H2309N385O421S11. The instability index: 42.58, the aliphatic index Aliphatic: 95.64, the total hydrophobicity: -0.162. PAP1 protein is hydrophilic, and there is a transmembrane region, about 42-79 amino acid fragments. No signal peptide. PAP1 gene belongs to the DREV1 transmethylase gene family. PAP1 mRNA was used to search for the similarity of GenBank database. The similarity between BLASTN1 gene and DREV1 gene was very high. When they were further paired with two sequences, the similarity was 80.8. In addition to the very poor similarity at the beginning and the end, the other parts had higher similarity with DREV1 CD4TCR 伪, 尾, 纬 multiple sequence alignment. There were several conserved loci. PAP1 was compared with the homologous genes of each species. The secondary structure of PAP1 protein was highly conserved. The secondary structure of PAP1 protein was mixed type 40%, 伪 helix 17%, 尾 folding 43%, other secondary structure. There are also N-glycosylation sites, protease C phosphorylation sites, casein protein kinase 鈪，

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