【摘要】：ProsaptideTX14是一種新型的神經(jīng)營養(yǎng)肽,由鞘脂激活蛋白原具有神經(jīng)營養(yǎng)活性的序列改造而成,鞘脂激活蛋白原或ProsaptideTX14可以與神經(jīng)細(xì)胞膜上的PT敏感的G受體結(jié)合,活化細(xì)胞外調(diào)節(jié)激酶(ERK),刺激神經(jīng)細(xì)胞中乙酰膽堿酯酶的活性,促進(jìn)神經(jīng)細(xì)胞的生長和分化。 由于鞘脂激活蛋白原為新發(fā)現(xiàn)的神經(jīng)營養(yǎng)因子,除了其神經(jīng)營養(yǎng)活性之外,在外圍組織的廣泛分布,提示ProsaptideTX14可能還有其它生物活性。因此,進(jìn)一步深入探討ProsaptideTX14的生物活性及其可能的分子機(jī)制,將為研究開發(fā)其潛在的臨床應(yīng)用價值奠定基礎(chǔ)。基因工程技術(shù)是制備短肽的一種行之有效的方法,我們將利用Prosaptide的氨基酸序列合成目的基因,以串聯(lián)連接方式得到多拷貝的目的片段,采用原核表達(dá)的方法高效的制備神經(jīng)營養(yǎng)肽NP14,進(jìn)而研究其生物活性。實(shí)驗(yàn)的主要內(nèi)容為: [目的] 采用基因工程的方法制備神經(jīng)營養(yǎng)肽NP14。 [方法] 1.NP14基因來源于ProsaptideTX14的14個氨基酸,它的第一個氨基酸由蘇氨酸改為絲氨酸,然后,通過PCR串聯(lián)成雙拷貝基因2NP,在小肽NP14兩端各引進(jìn)一個蛋氨酸,從而建立了蛋白裂解位點(diǎn)。 2.通過堿性磷酸酶的脫磷酸反應(yīng)處理的載體pUC18與雙拷貝基因2NP片段做平端連接,構(gòu)建了克隆載體pUC18—2NP。 3.通過藍(lán)白斑篩選得到的重組陽性克隆pUC18—2NP在大腸桿菌JM109中增殖后,經(jīng)質(zhì)粒的提取,EcoT14I的酶切,鑒定出陽性重
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：Q789

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本文編號：2337632