本文選題：GST-hDSL蛋白 + 抗GST-hDSL卵黃抗體�。� 參考：《上海交通大學(xué)》2007年碩士論文

【摘要】：Notch信號通路是被廣泛應(yīng)用的、進化高度保守的細(xì)胞分化調(diào)節(jié)系統(tǒng),在許多發(fā)育系統(tǒng)中對細(xì)胞的命運決定起著重要的調(diào)節(jié)作用[1]。Notch通路由Notch受體、Notch配體以及細(xì)胞內(nèi)效應(yīng)分子組成。近年來研究發(fā)現(xiàn),Notch受體廣泛表達于造血干/祖細(xì)胞以及成熟的血細(xì)胞,Notch配體表達于造血基質(zhì)細(xì)胞,提示Notch通路對造血系統(tǒng)的發(fā)生和發(fā)育具有調(diào)控作用[2-4]。在造血干細(xì)胞的培養(yǎng)中,通過激活Notch通路來維持干細(xì)胞的自我更新已經(jīng)取得了一定的進展,激活Notch信號通路有利于造血干/祖細(xì)胞的體外擴增。 Notch配體以膜蛋白和分泌蛋白的形式存在,其中胞外區(qū)含有一段在各個配體之間高度保守的結(jié)構(gòu)域,稱為DSL結(jié)構(gòu)域(DSL domain),它是配體與受體結(jié)合并激活受體所必需的結(jié)構(gòu)。hDelta-like-1(hDll-1)是人Notch配體之一,本實驗室通過對hDll-1蛋白結(jié)構(gòu)功能域的研究,選出了具有Notch受體激活功能的hDll-1最小功能蛋白序列,包括進化中的保守結(jié)構(gòu)區(qū)DSL和與之相鄰的N-端的50個氨基酸,命名為hDll-1NDSL(簡寫:hDSL)。本課題從克隆hDSL基因入手,構(gòu)建了重組質(zhì)粒pGEX-2T-hDSL。在大腸桿菌中誘導(dǎo)表達后,得到了帶有谷胱甘肽S轉(zhuǎn)移酶(glutathione transferase, GST)標(biāo)簽的GST-hDSL融合蛋白。重組蛋白主要以包涵體的形式存在,包涵體經(jīng)過變性復(fù)性、DEAE柱純化后,得到了高純度的重組蛋白。用此蛋白免疫蛋雞,獲得高純度的雞卵黃抗體IgY,為進一步研究GST-hDSL對造血干/祖細(xì)胞的擴增作用,奠定了良好的基礎(chǔ)。 本實驗的主要結(jié)果如下: 用PCR方法從重組質(zhì)粒pBlue-hDLL-1中克隆到目的基因片段hDSL,經(jīng)瓊脂糖凝膠電泳驗證約300bp大小,與理論值298bp基本符合。接著將此PCR產(chǎn)物和pGEX-2T空質(zhì)粒連接后,轉(zhuǎn)化到表達宿主菌DH5α中。經(jīng)菌落PCR、雙酶切鑒定以及測序證明:此hDSL序列大小和插入位置完全正確,至此pGEX-2T-hDSL重組質(zhì)粒構(gòu)建成功。 將成功構(gòu)建的重組質(zhì)粒pGEX-2T-hDSL轉(zhuǎn)化到大腸桿菌DH5α中,對蛋白誘導(dǎo)表達條件進行摸索后,確定最佳誘導(dǎo)條件為: 37℃、誘導(dǎo)4h、菌液含有青霉素100μg/mL和IPTG 1mM。經(jīng)SDS-PAGE電泳結(jié)果顯示:重組蛋白GST-hDSL主要以包涵體形式存在,分子量大小為37 kDa。 GST-hDSL蛋白大量表達:誘導(dǎo)1000mL菌液,經(jīng)超聲破菌、洗滌獲得初步純化的包涵體蛋白濕重0.5克。包涵體經(jīng)變性、復(fù)性和DEAE陰離子交換柱層析后獲得50mL目的蛋白溶液,Bradford蛋白定量法測得其濃度為0.8mg/mL,SDS-PAGE鑒定其純度可達95%。用此蛋白免疫蛋雞后,獲得高濃度的卵黃抗體IgY,免疫印跡能夠檢測到重組GST-hDSL蛋白,驗證了抗體特異性,為后期繼續(xù)研究GST-hDS蛋白的功能奠定了基礎(chǔ)。
[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 偽. 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 偽. After exploring the conditions of protein induction and expression, the optimal induction conditions were determined as follows: 37 鈩，

本文編號：1810947