利用基因工程制备神经营养肽NP14的研究

发布时间：2018-11-17 11:32

【摘要】：ProsaptideTX14是一种新型的神经营养肽,由鞘脂激活蛋白原具有神经营养活性的序列改造而成,鞘脂激活蛋白原或ProsaptideTX14可以与神经细胞膜上的PT敏感的G受体结合,活化细胞外调节激酶(ERK),刺激神经细胞中乙酰胆碱酯酶的活性,促进神经细胞的生长和分化。由于鞘脂激活蛋白原为新发现的神经营养因子,除了其神经营养活性之外,在外围组织的广泛分布,提示ProsaptideTX14可能还有其它生物活性。因此,进一步深入探讨ProsaptideTX14的生物活性及其可能的分子机制,将为研究开发其潜在的临床应用价值奠定基础。基因工程技术是制备短肽的一种行之有效的方法,我们将利用Prosaptide的氨基酸序列合成目的基因,以串联连接方式得到多拷贝的目的片段,采用原核表达的方法高效的制备神经营养肽NP14,进而研究其生物活性。实验的主要内容为: [目的] 采用基因工程的方法制备神经营养肽NP14。 [方法] 1.NP14基因来源于ProsaptideTX14的14个氨基酸,它的第一个氨基酸由苏氨酸改为丝氨酸,然后,通过PCR串联成双拷贝基因2NP,在小肽NP14两端各引进一个蛋氨酸,从而建立了蛋白裂解位点。 2.通过碱性磷酸酶的脱磷酸反应处理的载体pUC18与双拷贝基因2NP片段做平端连接,构建了克隆载体pUC18—2NP。 3.通过蓝白斑筛选得到的重组阳性克隆pUC18—2NP在大肠杆菌JM109中增殖后,经质粒的提取,EcoT14I的酶切,鉴定出阳性重
[Abstract]:ProsaptideTX14 is a new type of neurotrophic peptide, which is modified from the sequence of sphingomyelinogen with neurotrophic activity. Sphingomyelinogen or ProsaptideTX14 can bind to G receptor sensitive to PT on nerve cell membrane. Activation of extracellular kinase (ERK), stimulates the activity of acetylcholinesterase in neurons and promotes the growth and differentiation of nerve cells. Because sphingomyelinogen is a newly discovered neurotrophic factor, besides its neurotrophic activity, it is widely distributed in peripheral tissues, suggesting that ProsaptideTX14 may have other biological activities. Therefore, further study on the biological activity of ProsaptideTX14 and its possible molecular mechanism will lay a foundation for the research and development of its potential clinical application value. Genetic engineering is an effective method for the preparation of short peptides. We will synthesize the target gene by using the amino acid sequence of Prosaptide, and we will get multiple copies of the target fragment by tandem connection. The bioactivity of neurotrophic peptide NP14, was studied by prokaryotic expression. The main contents of the experiment were as follows: [objective] to prepare neurotrophic peptide NP14. by genetic engineering. [methods] the 1.NP14 gene was derived from 14 amino acids of ProsaptideTX14. Its first amino acid was changed from threonine to serine. The protein cleavage sites were established by introducing methionine at each end of the small peptide NP14. 2. The vector pUC18, which was treated by alkaline phosphatase dephosphorization, was connected with the double copy gene 2NP fragment to construct the clone vector pUC18-2NP.. 3. The recombinant positive clone pUC18-2NP was obtained by blue and white spot screening. After multiplication in Escherichia coli JM109, the positive weight was identified by extracting plasmid and digesting EcoT14I.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q789

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