人MGL的原核表达与鉴定及其真核表达载体构建

发布时间：2018-03-04 15:17

本文选题：凝集素　切入点：MGL　出处：《东北师范大学》2013年硕士论文　论文类型：学位论文

【摘要】：树突细胞（DC)是机体内重要的抗原递呈细胞，在机体的免疫应答中起关键作用。DC表面表达丰富的模式识别受体（PRRs)，主要包括Toll样受体（TLRs)和C-型凝集素受体（CLRs)。其中，，TLRs主要是通过与病原体的相互作用介导免疫激活，有利于病原体的清除，而CLRs则更倾向于介导机体的免疫耐受。近些年来的研究表明，CLR家族的一个成员，巨噬细胞半乳糖型凝集素（MGL)，参与了多种病原体的免疫逃逸。在本研究中，我们构建了4种MGL原核表达载体，用以选择MGL表达的最佳条件，其次，构建了2种MGL真核表达载体，用于不同类型细胞的转染。我们首先从外周血中分离单核细胞，经细胞因子诱导成为未成熟树突细胞（iDC)，然后提取iDC总RNA并将其逆转录为cDNA。经PCR获得MGL胞外基因片段，通过酶切连接构建了2种带有MGL胞外区的原核表达载体pET-28a(+)-MGL (胞外）和pGEX-6p-l-MGL (胞外）。将其在大肠杆菌中诱导表达，经蛋白表达形式分析发现，目的蛋白均以包涵体形式存在。为获得可溶性的目的蛋白，我们又构建了带有MGL全长基因片段的pET-28a(+)-MGL和pGEX-6p-l-MGL2种原核表达载体，通过在大肠杆菌中诱导表达和蛋白表达形式分析发现，只有pGEX-6p-l-MGL表达的目的蛋白大部分以可溶性形式存在。随后，将其转化菌体表达的上清蛋白，经GST琼脂糖亲和层析的方法进行纯化，得到GST-MGL融合蛋白，并通过WestemBlot验证即为目的蛋白。在原核表达载体构建的基础上，我们又构建带有MGL全长的2种真核表达载体pcDNA3.1(+)-MGL和pWPXLd-MGL。其中pcDNA3.1(+)-MGL适合于贴壁细胞的转染，而pWPXLd-MGL适合于悬浮细胞的转染。 MGL原核表达体系的构建对同类型凝集素的原核表达提供了设计思路，而且pGEX-6p-l-MGL原核表达载体表达的GST-MGL融合蛋白，可通过其标签GST的pulldown实验，研究与MGL共作用的受体和胞内信号转导蛋白。其真核表达载体可用于稳转细胞系的构建，对于研究依赖于MGL的细胞与细胞的识别以及MGL介导的信号通路均有重要意义。
[Abstract]:Dendritic cells (DC) are important antigen presenting cells in the body. It plays a key role in the immune response of the body. The pattern recognition receptor (PRRsN), which is abundant on the surface of DC, mainly includes the Toll like receptor (TLRs) and the C- type lectin receptor (C- lectin receptor), in which TLRs are mainly mediated by interaction with pathogens. In recent years, CLRs, a member of the macrophage galactose lectin, is involved in the immune escape of many pathogens. In this study, we constructed four MGL prokaryotic expression vectors to select the best conditions for MGL expression. Secondly, we constructed two MGL eukaryotic expression vectors for transfection of different types of cells. We first isolated monocytes from peripheral blood, induced by cytokines into immature dendritic cells, then extracted iDC total RNA and reverse transcripted it to cDNA.The extracellular gene fragment of MGL was obtained by PCR. Two prokaryotic expression vectors pET-28awith extracellular domain of MGL (pET-28a) and pGEX-6p-l-MGL (extracellular) were constructed by enzyme ligation. The expression of pET-28aand pGEX-6p-l-MGL were induced in Escherichia coli, and the expression forms of pET-28aand pGEX-6p-l-MGL were analyzed. In order to obtain the soluble target protein, we constructed the prokaryotic expression vector pET-28a (MGL and pGEX-6p-l-MGL2) with full-length MGL gene fragment. The results of induction expression and protein expression analysis in E. coli showed that only the target protein expressed by pGEX-6p-l-MGL existed in soluble form, and then the supernatant protein was transformed into the supernatant. The fusion protein of GST-MGL was purified by GST agarose affinity chromatography. The fusion protein was identified as the target protein by WestemBlot. The fusion protein was constructed on the basis of prokaryotic expression vector. We also constructed two eukaryotic expression vectors pcDNA3.1 (pWPXLd-MGLand pWPXLd-MGL) with full length of MGL, in which pcDNA3.1 (MGL is suitable for transfection of adherent cells and pWPXLd-MGL is suitable for transfection of suspension cells). The construction of MGL prokaryotic expression system provides a design idea for the prokaryotic expression of the same type lectin, and the GST-MGL fusion protein expressed by the pGEX-6p-l-MGL prokaryotic expression vector can be obtained through the pulldown experiment with the label GST. The eukaryotic expression vector can be used in the construction of stable cell line, and it is of great significance to study the recognition of MGL dependent cells and the signal pathway mediated by MGL.
【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：Q78;R392

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