GST-hDSL蛋白的原核表达和纯化及其鸡卵黄抗体的制备

发布时间：2018-04-27 13:36

  本文选题：GST-hDSL蛋白 + 抗GST-hDSL卵黄抗体　； 参考：《上海交通大学》2007年硕士论文

【摘要】：Notch信号通路是被广泛应用的、进化高度保守的细胞分化调节系统,在许多发育系统中对细胞的命运决定起着重要的调节作用[1]。Notch通路由Notch受体、Notch配体以及细胞内效应分子组成。近年来研究发现,Notch受体广泛表达于造血干/祖细胞以及成熟的血细胞,Notch配体表达于造血基质细胞,提示Notch通路对造血系统的发生和发育具有调控作用[2-4]。在造血干细胞的培养中,通过激活Notch通路来维持干细胞的自我更新已经取得了一定的进展,激活Notch信号通路有利于造血干/祖细胞的体外扩增。 Notch配体以膜蛋白和分泌蛋白的形式存在,其中胞外区含有一段在各个配体之间高度保守的结构域,称为DSL结构域(DSL domain),它是配体与受体结合并激活受体所必需的结构。hDelta-like-1(hDll-1)是人Notch配体之一,本实验室通过对hDll-1蛋白结构功能域的研究,选出了具有Notch受体激活功能的hDll-1最小功能蛋白序列,包括进化中的保守结构区DSL和与之相邻的N-端的50个氨基酸,命名为hDll-1NDSL(简写:hDSL)。本课题从克隆hDSL基因入手,构建了重组质粒pGEX-2T-hDSL。在大肠杆菌中诱导表达后,得到了带有谷胱甘肽S转移酶(glutathione transferase, GST)标签的GST-hDSL融合蛋白。重组蛋白主要以包涵体的形式存在,包涵体经过变性复性、DEAE柱纯化后,得到了高纯度的重组蛋白。用此蛋白免疫蛋鸡,获得高纯度的鸡卵黄抗体IgY,为进一步研究GST-hDSL对造血干/祖细胞的扩增作用,奠定了良好的基础。 本实验的主要结果如下: 用PCR方法从重组质粒pBlue-hDLL-1中克隆到目的基因片段hDSL,经琼脂糖凝胶电泳验证约300bp大小,与理论值298bp基本符合。接着将此PCR产物和pGEX-2T空质粒连接后,转化到表达宿主菌DH5α中。经菌落PCR、双酶切鉴定以及测序证明:此hDSL序列大小和插入位置完全正确,至此pGEX-2T-hDSL重组质粒构建成功。 将成功构建的重组质粒pGEX-2T-hDSL转化到大肠杆菌DH5α中,对蛋白诱导表达条件进行摸索后,确定最佳诱导条件为: 37℃、诱导4h、菌液含有青霉素100μg/mL和IPTG 1mM。经SDS-PAGE电泳结果显示:重组蛋白GST-hDSL主要以包涵体形式存在,分子量大小为37 kDa。 GST-hDSL蛋白大量表达:诱导1000mL菌液,经超声破菌、洗涤获得初步纯化的包涵体蛋白湿重0.5克。包涵体经变性、复性和DEAE阴离子交换柱层析后获得50mL目的蛋白溶液,Bradford蛋白定量法测得其浓度为0.8mg/mL,SDS-PAGE鉴定其纯度可达95%。用此蛋白免疫蛋鸡后,获得高浓度的卵黄抗体IgY,免疫印迹能够检测到重组GST-hDSL蛋白,验证了抗体特异性,为后期继续研究GST-hDS蛋白的功能奠定了基础。
[Abstract]:Notch signaling pathway is a widely used and highly conserved cellular differentiation regulatory system. In many developmental systems, it plays an important role in regulating the fate of cells [1] .Notch pathway consists of Notch receptor Notch ligand and intracellular effector molecules. In recent years, it has been found that the Notch receptor is widely expressed in hematopoietic stem / progenitor cells and mature blood cells, and that the ligand of Notch is expressed in hematopoietic stromal cells, suggesting that the Notch pathway plays a regulatory role in the genesis and development of hematopoietic system [2-4]. In the culture of hematopoietic stem cells, some progress has been made in maintaining self-renewal of stem cells by activating Notch pathway. Activation of Notch signaling pathway is beneficial to the expansion of hematopoietic stem / progenitor cells in vitro. Notch ligands exist in the form of membrane proteins and secretory proteins in which the extracellular domain contains a highly conserved domain between the various ligands. Called DSL domain, it is a necessary structure for ligand to bind to receptor and activate receptor. It is one of human Notch ligands. We studied the structural functional domain of hDll-1 protein in our laboratory. The minimal functional protein sequence of hDll-1 with activation function of Notch receptor was selected, including evolutional conserved domain DSL and 50 amino acids of adjacent N-terminal, named hDll-1NDSL. The recombinant plasmid pGEX-2T-hDSLwas constructed by cloning hDSL gene. After induced expression in E. coli, a glutathione transferase (GST) labeled GST-hDSL fusion protein was obtained. The recombinant protein mainly existed in the form of inclusion body. After denaturation and renaturation with DEAE column, the recombinant protein with high purity was obtained. High purity egg yolk antibody IgY was obtained by immunizing laying hens with this protein, which laid a good foundation for further study on the amplification effect of GST-hDSL on hematopoietic stem / progenitor cells. The main results of this experiment are as follows: The target gene fragment hDSL was cloned from recombinant plasmid pBlue-hDLL-1 by PCR method. The size of 300bp was confirmed by agarose gel electrophoresis, which was consistent with the theoretical value of 298bp. Then the PCR product was ligated with the empty plasmid of pGEX-2T and transformed into the host strain DH5 伪. The results of colony PCR, double digestion and sequencing showed that the hDSL sequence size and insertion position were correct, and the pGEX-2T-hDSL recombinant plasmid was successfully constructed. The recombinant plasmid pGEX-2T-hDSL was successfully transformed into Escherichia coli DH5 伪. After exploring the conditions of protein induction and expression, the optimal induction conditions were determined as follows: 37 鈩，

本文编号：1810947