原核高效可溶性表达载体的构建及初步应用

发布时间：2018-06-15 19:02

本文选题：表达载体 + 重组蛋白　；参考：《中国人民解放军军事医学科学院》2005年硕士论文

【摘要】：利用大肠杆菌生产多肽类药物具有成本低,周期短且不存在病毒致癌基因污染等优点,所以大肠杆菌是目前基因工程中细胞因子和多肽类药物生产的主要工程菌。但大多外源基因在大肠杆菌中表达时往往以包含体形式存在,这就造成复性的困难和成本的提高。原核生物表达系统缺乏真核细胞特有的加工后处理;高度还原环境大肠杆菌胞质使表达的重组蛋白往往不能形成正确的二硫键与完整的四级结构都使得重组蛋白的活性较低。因此如何获得高效可溶性并具有类似天然生物活性的重组蛋白是利用大肠杆菌进行基因工程技术的关键。大肠杆菌胞间质内的Dsb(二硫键氧化还原酶)家族使得胞间质内氧化电势较高为形成二硫键提供了有利条件。利用Dsb家族和目的蛋白融合表达具有让外源蛋白周质腔定位并辅助其正确形成二硫键和空间结构的功能。本课题基于以往的研究基础,利用Dsb蛋白家族的功能和特性构建了串联表达载体pET-SWG1-SWG2-NGF(神经生长因子β亚基)和pET-SWG1-SWG2-C_7+Fc(环七肽和Fc融合蛋白),并通过优化表达条件实现了融合蛋白SWG1-SWG2-NGF和SWG1-SWG2-C_7+Fc在大肠杆菌中高效可溶性表达。伴侣蛋白和目的蛋白之间凝血酶位点酶切和再次分离纯化后,获得了具有较高生物活性的目的蛋白NGFβ亚基和C_7+Fc。通过本课题研究,构建了高效可溶性表达载体,实现NGFβ亚基和C_7+Fc在原核高效可溶表达并具有良好生物活性。这为实现其它细胞因子或多肽类药物利用原核表达提供了模式。也为探讨原核生物的表达机制提供参考。
[Abstract]:The use of Escherichia coli to produce polypeptide drugs has the advantages of low cost, short period and no contamination of viral oncogenes, so Escherichia coli is the main engineering bacteria in the production of cytokines and polypeptide drugs in gene engineering. However, most of the foreign genes expressed in E. coli often exist in the form of inclusions, which leads to the difficulty of renaturation and the increase of cost. The expression system of prokaryotes lacks the unique post-processing of eukaryotic cells, and the cytoplasm of highly reduced Escherichia coli makes the expressed recombinant proteins often unable to form the correct disulfide bond and complete quaternary structure, which results in the low activity of the recombinant proteins. Therefore, how to obtain highly soluble recombinant protein with similar natural biological activity is the key to the genetic engineering of Escherichia coli. The Dsb (disulfide redox reductase) family in the interstitial of Escherichia coli provides favorable conditions for the formation of disulfide bonds. The fusion expression of DSB family and target protein has the function of localizing and assisting the formation of disulfide bonds and spatial structures of exogenous proteins. This subject is based on the previous research. A series expression vector pET-SWG1-SWG2-NGF (nerve growth factor 尾 subunit) and pET-SWG1-SWG2-C7Fc (cyclic heptapeptide and FC fusion protein) were constructed by using the function and characteristics of DSB protein family. The fusion proteins SWG1-SWG2-NGF and SWG1-SWG2-C7Fc were obtained by optimizing expression conditions. High efficient and soluble expression in bacteria. The target protein NGF 尾 subunit and C7Fc with high biological activity were obtained after digestion and purification of the thrombin site between chaperone protein and target protein. Through this study, a high efficient soluble expression vector was constructed to realize the expression of NGF 尾 subunit and C7Fc in prokaryotic cells, and the expression of NGF 尾 subunit and C7Fc in prokaryotic cells had good biological activity. This provides a model for the prokaryotic expression of other cytokines or polypeptide drugs. It also provides a reference for exploring the expression mechanism of prokaryotes.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q782

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