马立克氏病meq基因转染CEF后部分微卫星不稳定性和错配修复基因的研究

发布时间：2018-04-30 10:09

本文选题：马立克氏病meq基因 + 鸡胚成纤维细胞　；参考：《南京农业大学》2015年硕士论文

【摘要】：鸡马立克氏病(Marek's Disease,MD)是一种由血清型Ⅰ型马立克氏病毒(Marek's Disease Virus,MDV)引起的T淋巴细胞肿瘤性疾病,具有高度的传染性。MDV是唯一已知能急性转化的α疱疹病毒。MDV不仅可引起患鸡内脏T细胞淋巴瘤、瘫痪、失明和神经功能障碍,而且还可以引起较强的免疫抑制。微卫星(Microsatellite,MS)是一个位点特异的DNA序列,它们的核苷酸序列较短(重复单元为1-6个碱基对),核苷酸序列串联重复10-60次,其侧翼为独特的序列。在生物体基因组中,微卫星是有高丰度的特异性DNA标记。微卫星不稳定性(Microsatellite Instability,MSI)是由于复制错误(replication error,RER)引起的微卫星等位基因的增加或丢失,造成微卫星DNA长度发生改变,表现为(CA)n双核苷酸电泳迁移率的改变,出现与正常基因长度不同的等位基因条带。错配修复基因(Mismatch Repair,MMR)是切除修复未成对或错配的碱基并通过替换正确的DNA碱基对的一组基因。本试验对马立克氏病meq基因转染鸡胚成纤维细胞(CEF)后部分微卫星不稳定性和错配修复基因表达情况进行研究。研究目的是为马立克氏病meq基因的致瘤机制提供线索,试验分为以下三部分。第一部分是pVAX-1-meq真核表达载体的构建和在鸡胚成纤维细胞中表达验证。采用TA克隆将MEQ基因片段连接pMD 19-T Vector构建出pMD 19-T-meq质粒,pMD19-T-meq质粒和pVAX-1真核表达质粒采用EcorR I、Xho I限制性内切酶双酶切,将meq基因ORF克隆至pVAX-1质粒中,构建pVAX-1-meq真核表达质粒。转化DH5α感受态细胞,挑选阳性克隆采用pVAX-1质粒通用引物T7 primer和BGH reverse primer进行PCR验证和EcorR I、Xho I限制性内切酶双酶切验证。将pVAX-1-meq真核表达质粒用转染试剂转染至鸡胚成纤维细胞,并分别在转染后的24 h、48 h、72 h提取贴壁细胞总RNA,反转录成cDNA,根据meq基因mRNA设计引物MEQ mRNA F/R,以β-actin作为内参进行real-time PCR反应,检测出meq基因mRNA成功在细胞内表达。比较正常细胞和转染meq基因的细胞增殖,细胞凋亡,细胞周期的指标。转染meq基因后细胞凋亡率并未明显改变,细胞周期失控,G2期和S期阻滞,DNA复制过程受阻,MTT法检测发现细胞活性降低,说明转染MEQ基因至CEF细胞后并未使得CEF细胞发生转化,但会引起CEF的DNA合成受阻。试验第二部分是把构建好的pVAX-1-meq真核表达质粒用转染试剂转染至鸡胚成纤维细胞,同时设置空载体对照组、脂质体对照组和空白对照组,并分别在转染后的24h、48h、72h提取贴壁细胞总DNA;选取本实验室前期初步筛选的马立克氏病肿瘤中变化频率较高的微卫星标记,以此微卫星位点设计了 46对引物,以提取的DNA进行PCR扩增,采用变性聚丙烯酰胺凝胶电泳技术检测微卫星不稳定性,根据银染后的结果来看,并未发现实验组与对照组有明显的微卫星条带差异。试验第三部分是把构建好的pVAX-1-meq真核表达质粒用转染试剂转染至鸡胚成纤维细胞,同时设置空载体对照组、脂质体对照组和空白对照组,并分别在转染后的24h、48h、72h以β-actin作为内参进行real-time PCR反应,检测错配修复相关基因MLH1,MSH2和PMS1的mRNA表达情况,结果发现转染meq基因后24h,错配修复基因MLH1,MSH2和PMS1的相对表达量较各个对照组均有不同程度升高,而转染meq基因后48h、72h错配修复基因MLH1,MSH2和PMS1的相对表达量又低于对照组。这表明转染meq基因后24h错配修复系统开始发挥其功能,而在转染meq基因后48h和72h错配修复系统功能开始减弱或消失,这说明meq基因参与了错配修复相关基因的表达调控。
[Abstract]:Chicken Marek's disease (Marek's Disease, MD) is a T lymphocyte tumor caused by serotype I type Marek's Disease Virus (MDV). The highly infectious.MDV is the only known acute transformation of the alpha herpes virus.MDV not only can lead to chicken visceral T cell lymphoma, paralysis, blindness and nerve function. Microsatellite (MS) is a loci specific DNA sequence, their nucleotide sequences are short (repeat unit is 1-6 base pairs), nucleotide sequences are repeated 10-60 times in series, and their flanks are unique sequence. In the genome of the organism, microsatellites are highly specific. DNA markers. Microsatellite instability (Microsatellite Instability, MSI) is the increase or loss of microsatellite alleles due to the replication error (replication error, RER), resulting in a change in the length of microsatellite DNA, showing a change in the mobility of (CA) n double nucleoside acid electrophoresis, and a allelic strip that is different from the normal gene length. The mismatch repair gene (Mismatch Repair, MMR) is a group of genes excised to repair unpaired or mismatched bases and replace the correct DNA base pairs. This experiment studies the expression of microsatellite instability and mismatch repair genes in the posterior part of the Marek's disease meq gene transfected to chicken embryo fibroblasts (CEF). The purpose of this study is to study the purpose of this study. The tumor mechanism of the meq gene of Marek's disease provides clues. The experiment is divided into three parts. The first part is the construction of pVAX-1-meq eukaryotic expression vector and the expression in chicken embryo fibroblasts. The MEQ gene fragment is connected with pMD 19-T Vector to construct pMD 19-T-meq particles, pMD19-T-meq plasmids and pVAX-1 eukaryotic expression substance. EcorR I, Xho I restriction endonuclease double enzyme digestion, meq gene ORF was cloned into pVAX-1 plasmid, and pVAX-1-meq eukaryotic expression plasmid was constructed. DH5 alpha receptive cells were transformed and selected positive clones were verified by pVAX-1 plasmid T7 primer and purified restriction endonuclease verification. VAX-1-meq eukaryotic expression plasmid was transfected into chicken embryo fibroblast by transfection reagent, and the total RNA of the adherent cells was extracted at 24 h, 48 h and 72 h after transfection. The meq gene mRNA designed primers MEQ mRNA F/R. The proliferation, apoptosis, and cell cycle of normal cells and transfected mEq genes. The apoptosis rate of meq gene was not obviously changed, the cell cycle was out of control, the G2 phase and S block were blocked, the DNA replication process was blocked, and the MTT assay found that the cell activity decreased, indicating that the CEF cells were not transformed from the MEQ gene to CEF cells. The second part of the experiment was to transfect the constructed pVAX-1-meq eukaryotic expression plasmid to chicken embryo fibroblast by transfection reagent in the second part. At the same time, the empty carrier control group, the liposome control group and the blank control group were set up, and the total DNA of the adherent cells was extracted after the transfection of 24h, 48h, 72h, and the early stage of the laboratory was selected. 46 pairs of microsatellite markers with high frequency were selected for the preliminary screening of Marek's disease. The microsatellite loci were designed for 46 pairs of primers. The extracted PCR was amplified and the microsatellite instability was detected by denatured polyacrylamide gel electrophoresis. The results of silver staining showed that the experimental group was not obvious to the control group. The third part of the experiment was to transfect the constructed pVAX-1-meq eukaryotic expression plasmid into chicken embryo fibroblast by transfection reagent, and set up the empty carrier control group, the liposome control group and the blank control group. The 24h, 48h, and 72h were used as the internal reference for the real-time PCR reaction after the transfection, and the mismatch repair was detected. The mRNA expression of MLH1, MSH2 and PMS1 of the complex genes was found. The results showed that the relative expression of the mismatched repair gene MLH1, MSH2 and PMS1 was higher than that of the control group after transfection of the meq gene, while the meq gene was transfected and the relative expression of the 72h mismatch repair gene was lower than that of the control group. After gene 24h mismatch repair system began to function, the function of 48h and 72h mismatch repair system began to weaken or disappear after transfection of meq gene, which indicated that the meq gene was involved in the regulation of the expression of mismatch repair related genes.

【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31

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