發(fā)布時間：2018-04-30 10:09

  本文選題：馬立克氏病meq基因 + 雞胚成纖維細胞　； 參考：《南京農業(yè)大學》2015年碩士論文

【摘要】：雞馬立克氏病(Marek's Disease,MD)是一種由血清型Ⅰ型馬立克氏病毒(Marek's Disease Virus,MDV)引起的T淋巴細胞腫瘤性疾病,具有高度的傳染性。MDV是唯一已知能急性轉化的α皰疹病毒。MDV不僅可引起患雞內臟T細胞淋巴瘤、癱瘓、失明和神經功能障礙,而且還可以引起較強的免疫抑制。微衛(wèi)星(Microsatellite,MS)是一個位點特異的DNA序列,它們的核苷酸序列較短(重復單元為1-6個堿基對),核苷酸序列串聯(lián)重復10-60次,其側翼為獨特的序列。在生物體基因組中,微衛(wèi)星是有高豐度的特異性DNA標記。微衛(wèi)星不穩(wěn)定性(Microsatellite Instability,MSI)是由于復制錯誤(replication error,RER)引起的微衛(wèi)星等位基因的增加或丟失,造成微衛(wèi)星DNA長度發(fā)生改變,表現(xiàn)為(CA)n雙核苷酸電泳遷移率的改變,出現(xiàn)與正�；蜷L度不同的等位基因條帶。錯配修復基因(Mismatch Repair,MMR)是切除修復未成對或錯配的堿基并通過替換正確的DNA堿基對的一組基因。本試驗對馬立克氏病meq基因轉染雞胚成纖維細胞(CEF)后部分微衛(wèi)星不穩(wěn)定性和錯配修復基因表達情況進行研究。研究目的是為馬立克氏病meq基因的致瘤機制提供線索,試驗分為以下三部分。第一部分是pVAX-1-meq真核表達載體的構建和在雞胚成纖維細胞中表達驗證。采用TA克隆將MEQ基因片段連接pMD 19-T Vector構建出pMD 19-T-meq質粒,pMD19-T-meq質粒和pVAX-1真核表達質粒采用EcorR I、Xho I限制性內切酶雙酶切,將meq基因ORF克隆至pVAX-1質粒中,構建pVAX-1-meq真核表達質粒。轉化DH5α感受態(tài)細胞,挑選陽性克隆采用pVAX-1質粒通用引物T7 primer和BGH reverse primer進行PCR驗證和EcorR I、Xho I限制性內切酶雙酶切驗證。將pVAX-1-meq真核表達質粒用轉染試劑轉染至雞胚成纖維細胞,并分別在轉染后的24 h、48 h、72 h提取貼壁細胞總RNA,反轉錄成cDNA,根據meq基因mRNA設計引物MEQ mRNA F/R,以β-actin作為內參進行real-time PCR反應,檢測出meq基因mRNA成功在細胞內表達。比較正常細胞和轉染meq基因的細胞增殖,細胞凋亡,細胞周期的指標。轉染meq基因后細胞凋亡率并未明顯改變,細胞周期失控,G2期和S期阻滯,DNA復制過程受阻,MTT法檢測發(fā)現(xiàn)細胞活性降低,說明轉染MEQ基因至CEF細胞后并未使得CEF細胞發(fā)生轉化,但會引起CEF的DNA合成受阻。試驗第二部分是把構建好的pVAX-1-meq真核表達質粒用轉染試劑轉染至雞胚成纖維細胞,同時設置空載體對照組、脂質體對照組和空白對照組,并分別在轉染后的24h、48h、72h提取貼壁細胞總DNA;選取本實驗室前期初步篩選的馬立克氏病腫瘤中變化頻率較高的微衛(wèi)星標記,以此微衛(wèi)星位點設計了 46對引物,以提取的DNA進行PCR擴增,采用變性聚丙烯酰胺凝膠電泳技術檢測微衛(wèi)星不穩(wěn)定性,根據銀染后的結果來看,并未發(fā)現(xiàn)實驗組與對照組有明顯的微衛(wèi)星條帶差異。試驗第三部分是把構建好的pVAX-1-meq真核表達質粒用轉染試劑轉染至雞胚成纖維細胞,同時設置空載體對照組、脂質體對照組和空白對照組,并分別在轉染后的24h、48h、72h以β-actin作為內參進行real-time PCR反應,檢測錯配修復相關基因MLH1,MSH2和PMS1的mRNA表達情況,結果發(fā)現(xiàn)轉染meq基因后24h,錯配修復基因MLH1,MSH2和PMS1的相對表達量較各個對照組均有不同程度升高,而轉染meq基因后48h、72h錯配修復基因MLH1,MSH2和PMS1的相對表達量又低于對照組。這表明轉染meq基因后24h錯配修復系統(tǒng)開始發(fā)揮其功能,而在轉染meq基因后48h和72h錯配修復系統(tǒng)功能開始減弱或消失,這說明meq基因參與了錯配修復相關基因的表達調控。
[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.

【學位授予單位】：南京農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.31

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