本文選題：p53　切入點(diǎn)：下游基因　出處：《重慶大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】： p53基因是與人類腫瘤相關(guān)性最高的抑癌基因。前人研究表明, p53基因行使其生物學(xué)效應(yīng)主要是通過(guò)P53蛋白與特異基因組序列結(jié)合,激活下游基因轉(zhuǎn)錄、作用來(lái)實(shí)現(xiàn)的。因此,鑒定p53下游基因是了解p53基因調(diào)控網(wǎng)絡(luò)的關(guān)鍵。 本實(shí)驗(yàn)室在前期工作中建立攜帶野生型p53轉(zhuǎn)基因的U251-pTet-p53細(xì)胞系,在四環(huán)素應(yīng)答元件的控制下,誘導(dǎo)p53基因表達(dá)。通過(guò)差異顯示技術(shù),克隆到一個(gè)新的P53下游基因候選基因——PAP1基因。 本文構(gòu)建了pET28b-p53重組表達(dá)質(zhì)粒,將pET28b-p53轉(zhuǎn)化到大腸桿菌BL21(DE3),IPTG誘導(dǎo)表達(dá)P53蛋白。用地高辛標(biāo)記PAP1基因作為探針,與P53蛋白做凝膠滯留實(shí)驗(yàn),鑒定P53蛋白下游候選基因PAP1。然后綜合運(yùn)用生物信息學(xué)方法對(duì)PAP1基因進(jìn)行了基因結(jié)構(gòu)分析和功能分析。 研究結(jié)果顯示:(1)該基因序列能與野生型P53蛋白結(jié)合,與突變型P53蛋白幾乎不結(jié)合。因此PAP1基因是P53蛋白的下游基因,受P53蛋白調(diào)控。(2)PAP1基因定位于人類染色體16p12-13上,PAP1蛋白分子量為32.9KD,理論等電點(diǎn)pI為5.81,化學(xué)方程式為C1505H2309N385O421S11,半衰期為30小時(shí)左右,不穩(wěn)定指數(shù):42.58,脂肪族指數(shù)Aliphatic index:95.64,總平均疏水性:-0.162。PAP1蛋白為親水性蛋白,存在一個(gè)跨膜區(qū),大約在42-79氨基酸片段,沒(méi)有信號(hào)肽。(3)PAP1基因?qū)儆贒REV1轉(zhuǎn)甲基酶基因家族。PAP1mRNA對(duì)GenBank數(shù)據(jù)庫(kù)進(jìn)行相似性搜索-BLASTN,與DREV1基因的相似性很高,將它們進(jìn)一步做雙序列比對(duì)發(fā)現(xiàn)它們的相似性在80.8%,除了在開頭和結(jié)尾的相似性很差之外,其它部分相似性較高。PAP1與DREV1,CD4,TCRα、β、γ多序列比對(duì)時(shí),存在幾個(gè)保守的位點(diǎn)。PAP1與各個(gè)物種的同源基因的多序列比對(duì),它們的保守性很高。(4)PAP1蛋白的二級(jí)結(jié)構(gòu)為混合型,40%為α螺旋,17%為β折疊,43%為其它類型的二級(jí)結(jié)構(gòu);含有DREV1轉(zhuǎn)甲基酶結(jié)構(gòu)域,同時(shí)PAP1蛋白也存在N-糖基化位點(diǎn),蛋白酶C磷酸化位點(diǎn),干酪素蛋白激酶Ⅱ磷酸化位點(diǎn),N-十四�；稽c(diǎn),酪氨算鹽酸化作用位點(diǎn)。(5)與老鼠IGSF6基因同源性較高,可能是同源基因。IGSF6基因在老鼠胚胎發(fā)育中起著重要作用,依此推測(cè)PAP1基因也可能在人的胚胎發(fā)育中起著重要的作用。
[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 偽, 尾, 緯 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%, 偽 helix 17%, 尾 folding 43%, other secondary structure. There are also N-glycosylation sites, protease C phosphorylation sites, casein protein kinase 鈪，

本文編號(hào)：1640767