本文關鍵詞：人類Neuritin在原核細胞中的克隆、表達及相關生物學功能的研究　出處：《石河子大學》2006年碩士論文　論文類型：學位論文

  更多相關文章： Neuritin 原核表達 蛋白純化 活性測定

【摘要】： 神經(jīng)營養(yǎng)因子是一類促進神經(jīng)細胞存活、生長、分化的多肽分子,在神經(jīng)系統(tǒng)發(fā)育、分化和損傷修復過程中起著非常重要的作用。Neuritin是一種新近發(fā)現(xiàn)的與神經(jīng)可塑性相關的神經(jīng)營養(yǎng)因子。研究表明Neuritin在神經(jīng)再生的領域中具有較廣泛的生物學活性,可促進神經(jīng)突起的快速生長和分支,并參與調(diào)節(jié)神經(jīng)元突觸活動,因而對多種因素引起的神經(jīng)系統(tǒng)損傷后軸突和突起的生長具有潛在的治療與預防作用。由于Neuritin體內(nèi)含量低,提取與純化困難,難以滿足研究和臨床治療需要,因此以基因工程的方法制備重組人的Neuritin將是獲取Neuritin有效的手段。本論文就Neuritin在原核表達、純化及生物學活性測定等進行了研究,這為臨床上用基因治療神經(jīng)系統(tǒng)疾病奠定了實驗基礎。 本實驗是以neuritin cDNA為模板, PCR擴增neuritin基因后,克隆于原核表達載體pET32a,分別用NocI和NotI雙酶切鑒定。鑒定正確的重組子pET32-neuritin轉(zhuǎn)化大腸桿菌BL21,測序正確的陽性轉(zhuǎn)化子用IPTG誘導后獲得了融合Neuritin蛋白;SDS-PAGE分析表明:大腸桿菌表達菌株經(jīng)IPTG誘導后,可表達分子量為30kD的融合Neuritin蛋白質(zhì)并且在4小時時表達量最大;Western blot檢測表明該表達產(chǎn)物具有Neuritin免疫活性;經(jīng)Ni-NTA純化系統(tǒng)及尿素分步透析,表達蛋白得到了有效的純化和復性。最后,對重組Neuritin的活性進行鑒定。結(jié)果表明:與陰性和空白組對照后,大腸桿菌表達的融合的Neuritin在體外能促進培養(yǎng)的pc12細胞和雞胚背根神經(jīng)節(jié)神經(jīng)突起的生長并能延長它們的存活時間。 本研究首次報道了重組人Neuritin在大腸桿菌的表達以及在體外實驗中促進雞胚神經(jīng)節(jié)和pc12細胞突起的生長,這項工作為神經(jīng)營養(yǎng)因子Neuritin在神經(jīng)系統(tǒng)疾病的治療的應用中奠定了良好的基礎。
[Abstract]:Neurotrophic factor is a kind of promote nerve cell survival, growth, differentiation of polypeptide molecules, in nervous system development, differentiation and repair process plays a very important role in.Neuritin is a newly discovered associated with neural plasticity neurotrophic factor. The results show that Neuritin has extensive biological activity in nerve regeneration in the field, can promote the rapid growth and branching neurites, and regulate synapse activity, thus causing a variety of factors after nervous system injury of axons and neurite growth with treatment and prevention of the potential role of Neuritin in vivo. Because of low content of extraction and purification difficult, difficult to meet the needs of research and clinical treatment, so by the methods of gene engineering preparation of recombinant human Neuritin will get the Neuritin effective means. The expression of Neuritin in prokaryotic, pure and students The determination of physical activity was studied, which laid the experimental basis for the clinical use of gene therapy for the disease of the nervous system.
This experiment is based on Neuritin cDNA as template, PCR gene was amplified by Neuritin, cloned into prokaryotic expression vector pET32a, respectively NocI and NotI digestion. The correct identification of the recombinant pET32-neuritin was transformed into Escherichia coli BL21, sequencing of the positive transformants were obtained after induction by IPTG Neuritin fusion protein; SDS-PAGE analysis showed that: Escherichia coli strain induced by IPTG, the expression of the molecular weight of fusion protein 30kD and Neuritin in the 4 hour maximum expression; Western blot assay showed that the protein is active Neuritin; by Ni-NTA purification system and urea dialysis, the expressed protein was purified and refolded effectively. Finally, identification the activity of recombinant Neuritin. The results showed that: compared with the negative and blank control group, the fusion of Neuritin can promote PC12 cells and cultured in vitro expression of Chicken Escherichia coli The growth of neural protrusions in the dorsal root ganglion of the embryo can prolong their survival time.
In this study, we first reported the expression of recombinant human Neuritin in E.coli and the growth of the ganglion and PC12 cells in chicken embryos in vitro. This work laid a good foundation for the application of neurotrophic factor Neuritin in the treatment of nervous system diseases.

【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R346

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本文編號：1402973