人脑胶质原纤维酸性蛋白（GFAP）原核表达载体的构建及蛋白表达的鉴定

发布时间：2018-01-26 09:11

本文关键词： 胶质原纤维酸性蛋白质粒原核表达载体　出处：《第二军医大学》2009年硕士论文　论文类型：学位论文

【摘要】： 摘要:[目的]对胶质原纤维酸性蛋白(GFAP)进行基因克隆,构建原核表达质粒并加以鉴定。[方法]从人脑胶质瘤组织提取mRNA,采用RT-PCR的方法扩增GFAP序列并表达,然后与载体pGEX-4T-2连接,转化宿主菌BL21构建重组质粒,诱导表达后纯化,Western-blot鉴定。[结果]目的载体构建完成后,用双酶切、DNA测序与Western-blot的方法证实原核表达载体构建成功。[结论] pGEX-4T-2-GFAP原核表达载体的成功构建,为进一步研究出有效的基因工程疫苗奠定了基础。背景:胶质原纤维酸性蛋白(GFAP)在神经损伤的发生发展过程中具有重要作用。目的:对GFAP进行基因克隆,构建原核表达质粒并加以鉴定。设计、时间及地点:单一样本研究,于2008-09/11上海市长征医院神经外科实验室完成。材料:中间载体pGEM-T Easy质粒购于Promega公司,原核表达质粒pGEX-4T-2由上海市长征医院神经外科实验室保存。方法:从人脑胶质瘤组织提取mRNA,采用RT-PCR的方法扩增GFAP序列并表达,然后与载体pGEX-4T-2连接,转化宿主菌BL21构建重组质粒,诱导表达后纯化,Western-blot鉴定。主要观察指标:总RNA鉴定结果,PCR扩增产物分子质量的鉴定,重组表达载体的酶切鉴定,Western-blot鉴定。结果:目的载体构建完成后,用双酶切、DNA测序与Western-blot的方法证实原核表达载体构建成功。结论:pGEX-4T-2-GFAP原核表达载体的成功构建,为进一步研究出有效的基因工程疫苗奠定了基础。
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R341

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