本文選題：馬立克氏病meq基因 + 雞胚成纖維細(xì)胞�。� 參考：《南京農(nóng)業(yè)大學(xué)》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(xì)胞腫瘤性疾病,具有高度的傳染性。MDV是唯一已知能急性轉(zhuǎn)化的α皰疹病毒。MDV不僅可引起患雞內(nèi)臟T細(xì)胞淋巴瘤、癱瘓、失明和神經(jīng)功能障礙,而且還可以引起較強(qiáng)的免疫抑制。微衛(wèi)星(Microsatellite,MS)是一個(gè)位點(diǎn)特異的DNA序列,它們的核苷酸序列較短(重復(fù)單元為1-6個(gè)堿基對(duì)),核苷酸序列串聯(lián)重復(fù)10-60次,其側(cè)翼為獨(dú)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(biāo)記。微衛(wèi)星不穩(wěn)定性(Microsatellite Instability,MSI)是由于復(fù)制錯(cuò)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(zhǎng)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)與正常基因長(zhǎng)度不同的等位基因條帶。錯(cuò)配修復(fù)基因(Mismatch Repair,MMR)是切除修復(fù)未成對(duì)或錯(cuò)配的堿基并通過(guò)替換正確的DNA堿基對(duì)的一組基因。本試驗(yàn)對(duì)馬立克氏病meq基因轉(zhuǎn)染雞胚成纖維細(xì)胞(CEF)后部分微衛(wèi)星不穩(wěn)定性和錯(cuò)配修復(fù)基因表達(dá)情況進(jìn)行研究。研究目的是為馬立克氏病meq基因的致瘤機(jī)制提供線(xiàn)索,試驗(yàn)分為以下三部分。第一部分是pVAX-1-meq真核表達(dá)載體的構(gòu)建和在雞胚成纖維細(xì)胞中表達(dá)驗(yàn)證。采用TA克隆將MEQ基因片段連接pMD 19-T Vector構(gòu)建出pMD 19-T-meq質(zhì)粒,pMD19-T-meq質(zhì)粒和pVAX-1真核表達(dá)質(zhì)粒采用EcorR I、Xho I限制性?xún)?nèi)切酶雙酶切,將meq基因ORF克隆至pVAX-1質(zhì)粒中,構(gòu)建pVAX-1-meq真核表達(dá)質(zhì)粒。轉(zhuǎn)化DH5α感受態(tài)細(xì)胞,挑選陽(yáng)性克隆采用pVAX-1質(zhì)粒通用引物T7 primer和BGH reverse primer進(jìn)行PCR驗(yàn)證和EcorR I、Xho I限制性?xún)?nèi)切酶雙酶切驗(yàn)證。將pVAX-1-meq真核表達(dá)質(zhì)粒用轉(zhuǎn)染試劑轉(zhuǎn)染至雞胚成纖維細(xì)胞,并分別在轉(zhuǎn)染后的24 h、48 h、72 h提取貼壁細(xì)胞總RNA,反轉(zhuǎn)錄成cDNA,根據(jù)meq基因mRNA設(shè)計(jì)引物MEQ mRNA F/R,以β-actin作為內(nèi)參進(jìn)行real-time PCR反應(yīng),檢測(cè)出meq基因mRNA成功在細(xì)胞內(nèi)表達(dá)。比較正常細(xì)胞和轉(zhuǎn)染meq基因的細(xì)胞增殖,細(xì)胞凋亡,細(xì)胞周期的指標(biāo)。轉(zhuǎn)染meq基因后細(xì)胞凋亡率并未明顯改變,細(xì)胞周期失控,G2期和S期阻滯,DNA復(fù)制過(guò)程受阻,MTT法檢測(cè)發(fā)現(xiàn)細(xì)胞活性降低,說(shuō)明轉(zhuǎn)染MEQ基因至CEF細(xì)胞后并未使得CEF細(xì)胞發(fā)生轉(zhuǎn)化,但會(huì)引起CEF的DNA合成受阻。試驗(yàn)第二部分是把構(gòu)建好的pVAX-1-meq真核表達(dá)質(zhì)粒用轉(zhuǎn)染試劑轉(zhuǎn)染至雞胚成纖維細(xì)胞,同時(shí)設(shè)置空載體對(duì)照組、脂質(zhì)體對(duì)照組和空白對(duì)照組,并分別在轉(zhuǎn)染后的24h、48h、72h提取貼壁細(xì)胞總DNA;選取本實(shí)驗(yàn)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(biāo)記,以此微衛(wèi)星位點(diǎn)設(shè)計(jì)了 46對(duì)引物,以提取的DNA進(jìn)行PCR擴(kuò)增,采用變性聚丙烯酰胺凝膠電泳技術(shù)檢測(cè)微衛(wèi)星不穩(wěn)定性,根據(jù)銀染后的結(jié)果來(lái)看,并未發(fā)現(xiàn)實(shí)驗(yàn)組與對(duì)照組有明顯的微衛(wèi)星條帶差異。試驗(yàn)第三部分是把構(gòu)建好的pVAX-1-meq真核表達(dá)質(zhì)粒用轉(zhuǎn)染試劑轉(zhuǎn)染至雞胚成纖維細(xì)胞,同時(shí)設(shè)置空載體對(duì)照組、脂質(zhì)體對(duì)照組和空白對(duì)照組,并分別在轉(zhuǎn)染后的24h、48h、72h以β-actin作為內(nèi)參進(jìn)行real-time PCR反應(yīng),檢測(cè)錯(cuò)配修復(fù)相關(guān)基因MLH1,MSH2和PMS1的mRNA表達(dá)情況,結(jié)果發(fā)現(xiàn)轉(zhuǎn)染meq基因后24h,錯(cuò)配修復(fù)基因MLH1,MSH2和PMS1的相對(duì)表達(dá)量較各個(gè)對(duì)照組均有不同程度升高,而轉(zhuǎn)染meq基因后48h、72h錯(cuò)配修復(fù)基因MLH1,MSH2和PMS1的相對(duì)表達(dá)量又低于對(duì)照組。這表明轉(zhuǎn)染meq基因后24h錯(cuò)配修復(fù)系統(tǒng)開(kāi)始發(fā)揮其功能,而在轉(zhuǎn)染meq基因后48h和72h錯(cuò)配修復(fù)系統(tǒng)功能開(kāi)始減弱或消失,這說(shuō)明meq基因參與了錯(cuò)配修復(fù)相關(guān)基因的表達(dá)調(diào)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(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S858.31

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 付煜;杜小燕;;微衛(wèi)星不穩(wěn)定性與腫瘤的研究進(jìn)展[J];腫瘤學(xué)雜志;2012年01期

2 陳欣虹;錢(qián)琨;郁川;田野;秦愛(ài)建;;雞內(nèi)參基因β-actin和GAPDH實(shí)時(shí)定量PCR重組質(zhì)粒標(biāo)準(zhǔn)品的構(gòu)建[J];中國(guó)家禽;2011年10期

3 鐘智偉;潘琳;龍香娥;樂(lè)燕萍;龔朝輝;;DNA錯(cuò)配修復(fù)與肺癌[J];中國(guó)細(xì)胞生物學(xué)學(xué)報(bào);2011年02期

4 藍(lán)響;;馬立克氏病病毒的分子生物學(xué)特性和檢測(cè)及疫苗研究進(jìn)展[J];家禽科學(xué);2009年11期

5 龔振華;李葳;劉國(guó)慶;;淺談馬立克氏病防治研究進(jìn)展[J];中國(guó)動(dòng)物檢疫;2009年08期

6 褚秀玲;蘇建青;付本懂;王魯;王春元;申海青;韋旭斌;;雞馬立克病研究進(jìn)展[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2009年01期

7 韋平;;馬立克氏病研究的最新動(dòng)態(tài)——第八屆國(guó)際馬立克氏病研討會(huì)概況[J];中國(guó)家禽;2008年17期

8 田正康;石銀亮;唐玉章;叢永博;曹育明;;影響雞馬立克氏病免疫效果的因素及對(duì)策[J];畜牧與飼料科學(xué);2007年06期

9 刁秀國(guó);成子強(qiáng);朱國(guó);王桂花;孟祥凱;高婷婷;;肉種雞馬立克氏病的動(dòng)態(tài)病理組織學(xué)研究[J];西南農(nóng)業(yè)學(xué)報(bào);2007年04期

10 龔新勇;陳紅;李永明;溫貴蘭;;蛋雞J亞群禽白血病與馬立克氏病混合感染的診斷[J];中國(guó)家禽;2007年15期

相關(guān)博士學(xué)位論文 前1條

1 張志;我國(guó)近年來(lái)馬立克氏病病毒野毒株分子流行病學(xué)和生物學(xué)特性的研究[D];山東農(nóng)業(yè)大學(xué);2004年

相關(guān)碩士學(xué)位論文 前1條

1 張超;馬立克氏病患雞中錯(cuò)配修復(fù)基因PMS1的初步研究[D];南京農(nóng)業(yè)大學(xué);2011年

，

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