人类Neuritin在原核细胞中的克

发布时间：2018-01-09 23:25

本文关键词：人类Neuritin在原核细胞中的克隆、表达及相关生物学功能的研究　出处：《石河子大学》2006年硕士论文　论文类型：学位论文

【摘要】： 神经营养因子是一类促进神经细胞存活、生长、分化的多肽分子,在神经系统发育、分化和损伤修复过程中起着非常重要的作用。Neuritin是一种新近发现的与神经可塑性相关的神经营养因子。研究表明Neuritin在神经再生的领域中具有较广泛的生物学活性,可促进神经突起的快速生长和分支,并参与调节神经元突触活动,因而对多种因素引起的神经系统损伤后轴突和突起的生长具有潜在的治疗与预防作用。由于Neuritin体内含量低,提取与纯化困难,难以满足研究和临床治疗需要,因此以基因工程的方法制备重组人的Neuritin将是获取Neuritin有效的手段。本论文就Neuritin在原核表达、纯化及生物学活性测定等进行了研究,这为临床上用基因治疗神经系统疾病奠定了实验基础。本实验是以neuritin cDNA为模板, PCR扩增neuritin基因后,克隆于原核表达载体pET32a,分别用NocI和NotI双酶切鉴定。鉴定正确的重组子pET32-neuritin转化大肠杆菌BL21,测序正确的阳性转化子用IPTG诱导后获得了融合Neuritin蛋白;SDS-PAGE分析表明:大肠杆菌表达菌株经IPTG诱导后,可表达分子量为30kD的融合Neuritin蛋白质并且在4小时时表达量最大;Western blot检测表明该表达产物具有Neuritin免疫活性;经Ni-NTA纯化系统及尿素分步透析,表达蛋白得到了有效的纯化和复性。最后,对重组Neuritin的活性进行鉴定。结果表明:与阴性和空白组对照后,大肠杆菌表达的融合的Neuritin在体外能促进培养的pc12细胞和鸡胚背根神经节神经突起的生长并能延长它们的存活时间。本研究首次报道了重组人Neuritin在大肠杆菌的表达以及在体外实验中促进鸡胚神经节和pc12细胞突起的生长,这项工作为神经营养因子Neuritin在神经系统疾病的治疗的应用中奠定了良好的基础。
[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.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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