人神经营养素-4的原核表达与纯化

发布时间：2018-08-24 16:26

【摘要】：研究目的:神经营养素家族是一类由靶组织分泌,具有促进和维持神经细胞生存、生长、分化等功能的特异性蛋白质或多肽类分子。神经营养素-4是第四个被发现的神经营养素家族成员,研究表明神经营养素-4对鸡胚背根神经节的感觉神经元的存活和生长有促进作用。但人神经营养素-4(hNT-4)在机体内含量非常低,因而限制了对它的研究。本实验的目的就是要通过克隆hNT-4成熟蛋白基因片段,并使其在高效原核表达载体中大量表达和纯化,得到高纯度蛋白,为对hNT-4的进一步研究打下基础。研究方法:以正常人外周血淋巴细胞基因组DNA为模板,通过常规PCR扩增出hNT-4成熟蛋白基因片段。再通过双限制性内切酶进行酶切,连接入高效原核表达载体pET32a中,重组质粒pET-NT4在大肠杆菌DH5α中进行克隆后,转化入其表达宿主菌BL21(DE3)中进行表达。对重组菌表达条件进行优化后,对重组菌进行扩大培养,使其诱导表达出带有hNT-4成熟蛋白的融合蛋白。超声波破碎菌体后,分离出包涵体后,将包涵体溶于8M尿素后,通过Ni~(2+)-鏊合亲和层析柱进行纯化。研究结果:克隆得到的hNT-4成熟蛋白基因与GeneBank报道的序列除了有一个碱基不同以外,其余的都完全相同。重组质粒pET-NT4构建正确,在表达宿主菌中表达出了预期的融合蛋白。融合蛋白主要存在于包涵体中,包涵体溶于8M尿素后,用Ni~(2+)-鏊合亲和层析柱对溶解液进行纯化,在0.15M咪唑洗涤液中得到的大部分融合蛋白,并且纯度达到94%。研究结论:成功扩增出人神经营养素-4成熟蛋白基因片段,构建了原核表达重组质粒pET-NT4,表达得到人神经营养素-4成熟蛋白和硫氧还原蛋白的融合蛋白,纯化后得到了高纯度(94%)的融合蛋白。
[Abstract]:Objective: the neurotrophin family is a class of specific proteins or polypeptides secreted by target tissues which can promote and maintain the survival, growth and differentiation of nerve cells. Neurotrophin-4 is the fourth member of the neurotrophin family which has been found to promote the survival and growth of sensory neurons in the dorsal root ganglion of chicken embryo. However, human neurotrophin-4 (hNT-4) is very low in the body, which limits its research. The purpose of this experiment is to clone the hNT-4 mature protein gene fragment, express and purify it in a large number in prokaryotic expression vector, and obtain the high purity protein, which will lay a foundation for the further study of hNT-4. Methods: genomic DNA of human peripheral blood lymphocytes was used as template and hNT-4 mature protein gene fragment was amplified by routine PCR. The recombinant plasmid pET-NT4 was cloned into Escherichia coli DH5 伪 and transformed into the expression host strain BL21 (DE3). After optimizing the expression conditions of the recombinant bacteria, the recombinant bacteria were expanded to express the fusion protein with hNT-4 mature protein. The inclusion bodies were separated by ultrasonic wave and dissolved in 8M urea. The inclusion bodies were purified by Ni~ (2)-chelate affinity chromatography. The results showed that the cloned hNT-4 mature protein gene was identical to the sequence reported by GeneBank except for one base. The recombinant plasmid pET-NT4 was constructed correctly and the expected fusion protein was expressed in the expressed host strain. The fusion protein was mainly present in the inclusion body. After the inclusion body was dissolved in 8M urea, the soluble solution was purified by Ni~ (2) -chelate affinity chromatography. Most of the fusion proteins were obtained in the 0.15m imidazole washing solution, and the purity of the fusion protein reached 94%. Conclusion: the human neurotrophin-4 mature protein gene fragment was amplified successfully, and the fusion protein of human neurotrophin-4 mature protein and thioxy-reducing protein was obtained by prokaryotic expression of recombinant plasmid pET-NT4,. After purification, a high purity (94%) fusion protein was obtained.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R341

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