發(fā)布時間：2018-01-18 12:32

  本文關(guān)鍵詞：重組Mash1慢病毒表達(dá)載體的構(gòu)建及表達(dá)　出處：《鄭州大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Mash1 bHLH 基因轉(zhuǎn)染 基因表達(dá) RT-PCR

【摘要】：背景和目的： bHLH(basic helix-loop-helix，堿性螺旋-環(huán)-螺旋)轉(zhuǎn)錄調(diào)控因子是發(fā)育過程中轉(zhuǎn)錄網(wǎng)絡(luò)的重要組成部分，，廣泛參與神經(jīng)和肌肉發(fā)生、細(xì)胞增殖分化、細(xì)胞譜系的決定和性別決定等基本生理過程。Mash1(mammalian achaete-scute homolog 1)基因?qū)儆赽HLH轉(zhuǎn)錄調(diào)控因子家族成員之一，在神經(jīng)發(fā)生、神經(jīng)發(fā)育和神經(jīng)分化過程中發(fā)揮重要的調(diào)控作用。Mash1基因功能缺失導(dǎo)致神經(jīng)發(fā)育異常、神經(jīng)元分化減少及特定亞型神經(jīng)元缺失；而其過表達(dá)則可以促進(jìn)P19細(xì)胞、神經(jīng)干細(xì)胞和胚胎干細(xì)胞向神經(jīng)細(xì)胞分化。由于Mash1基因的過表達(dá)研究多數(shù)應(yīng)用的是質(zhì)粒型表達(dá)載體，轉(zhuǎn)染效率較低且不能穩(wěn)定表達(dá)，如采用合適的載體將Mash1基因?qū)敫杉?xì)胞或體內(nèi)或?qū)⑥D(zhuǎn)染Mash1基因的靶細(xì)胞植入損傷局部，使其在損傷部位持續(xù)分泌Mash1蛋白，必將有利于神經(jīng)分化和神經(jīng)損傷的修復(fù)。因此，為進(jìn)一步探討Mash1基因在神經(jīng)發(fā)育和干細(xì)胞神經(jīng)分化中的調(diào)控作用及為基因治療神經(jīng)系統(tǒng)疾病奠定基礎(chǔ)，我們擬構(gòu)建重組Mash1基因慢病毒表達(dá)載體。 實驗方法： 1．Mash1cDNA克隆及序列分析 從小鼠胚胎中提取總RNA，逆轉(zhuǎn)錄為cDNA，PCR擴(kuò)增獲得小鼠Mash1基因，回收純化；以T4連接酶連接Mash1基因片段和pGEM-T載體，轉(zhuǎn)化感受態(tài)細(xì)菌JM109后經(jīng)藍(lán)白篩選和以T7／SP6為引物PCR擴(kuò)增篩選鑒定重組克隆；小提重組克隆質(zhì)粒DNA，應(yīng)用限制性內(nèi)切酶BamH Ⅰ和Xhol Ⅰ對其進(jìn)一步酶切鑒定陽性克�。粚﹁b定出的陽性重組質(zhì)粒pGEM-T-Mash1進(jìn)行基因測序。
[Abstract]:Background and purpose: BHLH(basic helix-loop-helix (basic helix-loop-helix) transcription regulator is an important part of transcription network during development. Extensive involvement in neurogenesis and musculogenesis, cell proliferation and differentiation. Basic physiological processes, such as cell lineage determination and sex determination. Mash1 mammalian achaete-scute homolog 1). Genes belong to the family of bHLH transcription regulators. Mash1 gene dysfunction in neurogenesis, neurogenesis and neural differentiation. The absence of Mash1 gene leads to abnormal neural development, reduced neuronal differentiation and specific subtype neuronal deletion. However, overexpression can promote the differentiation of P19 cells, neural stem cells and embryonic stem cells into neural cells. Because of the overexpression of Mash1 gene, most of them are plasmid expression vectors. Transfection efficiency is low and can not be stably expressed, such as the appropriate vector to transfer Mash1 gene into stem cells or in vivo or target cells transfected with Mash1 gene implanted into the local injury. The continuous secretion of Mash1 protein in the injured site will be conducive to nerve differentiation and nerve injury repair. To further explore the role of Mash1 gene in neural development and neural differentiation of stem cells and to lay a foundation for gene therapy of nervous system diseases. We intend to construct the recombinant Mash1 gene lentivirus expression vector. Experimental methods: 1. Mash1 cDNA was cloned and sequenced. Total RNAs were extracted from mouse embryos. Mouse Mash1 gene was amplified by reverse transcription-PCR with cDNA-DNA, and the mouse Mash1 gene was recovered and purified. Mash1 gene fragment and pGEM-T vector were ligated with T4 ligase. The recombinant clones were identified by blue and white screening and PCR amplification and screening using T7 / SP6 as primer. The recombinant plasmid DNA was isolated and identified by restriction endonuclease BamH 鈪

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