本文關(guān)鍵詞： 膠質(zhì)原纖維酸性蛋白 質(zhì)粒 原核表達載體　出處：《第二軍醫(yī)大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 摘要:[目的]對膠質(zhì)原纖維酸性蛋白(GFAP)進行基因克隆,構(gòu)建原核表達質(zhì)粒并加以鑒定。[方法]從人腦膠質(zhì)瘤組織提取mRNA,采用RT-PCR的方法擴增GFAP序列并表達,然后與載體pGEX-4T-2連接,轉(zhuǎn)化宿主菌BL21構(gòu)建重組質(zhì)粒,誘導(dǎo)表達后純化,Western-blot鑒定。[結(jié)果]目的載體構(gòu)建完成后,用雙酶切、DNA測序與Western-blot的方法證實原核表達載體構(gòu)建成功。[結(jié)論] pGEX-4T-2-GFAP原核表達載體的成功構(gòu)建,為進一步研究出有效的基因工程疫苗奠定了基礎(chǔ)。 背景:膠質(zhì)原纖維酸性蛋白(GFAP)在神經(jīng)損傷的發(fā)生發(fā)展過程中具有重要作用。 目的:對GFAP進行基因克隆,構(gòu)建原核表達質(zhì)粒并加以鑒定。 設(shè)計、時間及地點:單一樣本研究,于2008-09/11上海市長征醫(yī)院神經(jīng)外科實驗室完成。 材料:中間載體pGEM-T Easy質(zhì)粒購于Promega公司,原核表達質(zhì)粒pGEX-4T-2由上海市長征醫(yī)院神經(jīng)外科實驗室保存。 方法:從人腦膠質(zhì)瘤組織提取mRNA,采用RT-PCR的方法擴增GFAP序列并表達,然后與載體pGEX-4T-2連接,轉(zhuǎn)化宿主菌BL21構(gòu)建重組質(zhì)粒,誘導(dǎo)表達后純化,Western-blot鑒定。 主要觀察指標:總RNA鑒定結(jié)果,PCR擴增產(chǎn)物分子質(zhì)量的鑒定,重組表達載體的酶切鑒定,Western-blot鑒定。 結(jié)果:目的載體構(gòu)建完成后,用雙酶切、DNA測序與Western-blot的方法證實原核表達載體構(gòu)建成功。 結(jié)論:pGEX-4T-2-GFAP原核表達載體的成功構(gòu)建,為進一步研究出有效的基因工程疫苗奠定了基礎(chǔ)。
[Abstract]:Abstract:. [Objective: to clone the gene of glial fibrillary acidic protein (GFAP), construct the prokaryotic expression plasmid and identify it. [Methods: mRNAs were extracted from human glioma tissues, GFAP sequence was amplified and expressed by RT-PCR, and then ligated with vector pGEX-4T-2. The recombinant plasmid was constructed by transforming the host strain BL21, and then purified by Western-blot. [Results: after the construction of the vector, the construction of prokaryotic expression vector was confirmed by double enzyme digestion and Western-blot. [Conclusion the successful construction of prokaryotic expression vector of pGEX-4T-2-GFAP lays a foundation for the further study of effective genetic engineering vaccine. Background: glial fibrillary acidic protein (GFAP) plays an important role in the development of nerve injury. Objective: to clone GFAP gene, construct prokaryotic expression plasmid and identify it. Design, time and place: this single study was completed in the Neurosurgery Laboratory of Shanghai long March Hospital, 2008-09 / 11. Materials: the intermediate vector pGEM-T Easy plasmid was purchased from Promega Company. The prokaryotic expression plasmid pGEX-4T-2 was preserved by neurosurgery laboratory of Shanghai Changzheng Hospital. Methods: mRNAs were extracted from human glioma tissues. GFAP sequence was amplified and expressed by RT-PCR, then ligated with vector pGEX-4T-2. The recombinant plasmid was constructed by transforming the host strain BL21, and then purified by Western-blot. Main outcome measures: identification of molecular weight of amplified products from total RNA and Western blot analysis of recombinant expression vector by restriction endonuclease digestion. Results: after the construction of the vector, the construction of prokaryotic expression vector was confirmed by double enzyme digestion and Western-blot. Conclusion the successful construction of the prokaryotic expression vector of pGEX-4T-2-GFAP lays a foundation for the further development of an effective genetic engineering vaccine.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R341

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