IKK-a-pEGFP、TRAF1-pDsRed、EGFP-pDsRed融合蛋白重组体的构建和表达

发布时间：2018-07-10 03:52

本文选题：EGFP + DsRed　；参考：《华中科技大学》2006年硕士论文

【摘要】： 肿瘤坏死因子-α(tumor necrosis factor-α)主要由活化的T细胞、单核/巨噬细胞、NK细胞产生并分泌,具有多种不同的生物学功能。它以两种生物活性形式存在,即26kD的跨膜型TNF-α(transmembran TNF-α,TM-TNF-α)和17kD的分泌型TNF-α(secreted TNF-α, S-TNF-α)。TM-TNF-α是S-TNF-α的前体,在TNF-α转换酶(TACE)的作用下,其胞外段被切割,脱落成S-TNF-α。近年来研究发现,TM-TNF-α和其受体结合后,不仅可以启动受体后的信号传导,即正向信号,而且能向自身所在的细胞传递信号,即“反向信号”(reverse signaling),并引起相应的生物学效应。本室的前期实验通过免疫共沉淀技术证实IKK-α和TRAF1分别可以与TM-TNF-α共沉淀下来,提示这两种信号分子有可能以直接或间接的方式与TM-TNF-α的胞浆段结合,传递反向信号。本实验主要通过基因工程技术从细胞中获取IKK-α和TRAF1全长编码基因,分别与可以发生荧光共振能量转移(FRET)的荧光蛋白对构建成融合蛋白,同时构建荧光共振能量转移检测用的阳性质粒,为通过荧光共振能量转移技术进一步研究它们在活体细胞内相互之间的作用奠定基础。本研究主要结果如下: 一. IKK-α-pEGFP、TRAF1-pDsRed、EGFP-pDsRed构建、克隆及鉴定 1.重组体的构建和克隆:提取Raji细胞总RNA进行逆转录,以cDNA为模板,用PCR扩增IKK-α和TRAF1目的基因;通过巢式PCR进行鉴定,以确认扩增片段的特异性。同时以pIRES-EGFP质粒为模板,经PCR扩增得到EGFP目的基因片断。以Xho I和EcoR I双酶切pEGFP-N3质粒和IKK-α目的基因,以EcoR I和SacII双酶切pDsRed-N1质粒和TRAF1及EGFP目的基因,并用T4 DNA连接酶连接,获得IKK-α-pEGFP、TRAF1-pDsRed及EGFP-pDsRed的重组体,然后将连接产物转化DH5α感受态细菌,挑选阳性克隆。 2.阳性克隆的鉴定:以菌落PCR初步筛选阳性克隆,再用上述限制性内切酶双酶切鉴定,结果显示三种重组体经酶切分别能得到2.2Kb、1.3Kb和720bp的目的片断,与连接的目的基因大小一致。进一步DNA测序分析,证实三个重组体均各包含IKK-α、TRAF1和EGFP的编码区,没有突变和移码。提示IKK-α-pEGFP、TRAF1-pDsRed及EGFP-pDsRed三个重组体已经构建成功。二. IKK-α-pEGFP、TRAF1-pDsRed、EGFP-pDsRed三个重组体在真核细胞表达与鉴定将三种重组体分别转染COS7细胞,可见IKK-α-pEGFP融合蛋白(绿色荧光)弥散分布在胞浆内,TRAF1-pDsRed融合蛋白(红色荧光)则局限在细胞亚区。而表达EGFP-pDsRed融合蛋白既能呈现绿色荧光,又可呈现红色荧光,主要分布在胞浆内。提示IKK-α-pEGFP、TRAF1-pDsRed、EGFP-pDsRed三种融合蛋白均在真核细胞中被成功表达。本实验成功地构建了IKK-α-pEGFP、TRAF1-pDsRed、EGFP-pDsRed三种重组体,且在真核细胞中得到表达,并证实IKK-α和TRAF1两种分子的胞内定位。为进一步用FRET技术研究两分子间及与TM-TNF-α胞内段之间的相互作用奠定基础。EGFP-pDsRed阳性质粒的构建也为FRET检测提供阳性参照。
[Abstract]:Tumor necrosis factor - alpha (tumor necrosis factor- alpha) is mainly produced and secreted by activated T cells, mononuclear / macrophages and NK cells. It has a variety of different biological functions. It exists in two forms of biological activity, namely, 26kD's transmembrane TNF- alpha (transmembran TNF- a, TM-TNF- a) and 17kD secretory alpha alpha (alpha). .TM-TNF- alpha is a precursor of S-TNF- alpha. Under the action of TNF- alpha converting enzyme (TACE), its extracellular segment is cut off and shedding into S-TNF- alpha.
In recent years, it has been found that the binding of TM-TNF- alpha to its receptor can not only initiate the signal transduction after the receptor, namely the positive signal, but also transmit the signal to the cell in its own, that is the "reverse signaling", and causes the corresponding biological effects.
The previous experiments in the laboratory confirmed that IKK- alpha and TRAF1 could be co precipitated with TM-TNF- alpha by immunoprecipitation technique, suggesting that these two signal molecules may be combined with the cytoplasm of TM-TNF- alpha in a direct or indirect way and transmit the reverse signal.
In this experiment, IKK- alpha and TRAF1 full-length encoding genes were obtained from the cells by genetic engineering technology, and the fusion protein was constructed with fluorescence resonance energy transfer (FRET) fluorescent protein, respectively, and the positive plasmids used for fluorescence resonance energy transfer detection were constructed, which was further studied by fluorescence resonance energy transfer technology. The results are as follows:
Construction, cloning and identification of IKK- alpha -pEGFP, TRAF1-pDsRed and EGFP-pDsRed
1. the construction and cloning of the recombinant: extracting the total RNA of Raji cells for reverse transcription, using cDNA as the template, amplification of IKK- alpha and TRAF1 target genes with PCR, identification by nested PCR to confirm the specificity of the amplified fragment. At the same time, pIRES-EGFP plasmid was used as a template to amplify the fragment of EGFP target gene. The grain and IKK- alpha target genes, using EcoR I and SacII double enzymes to cut pDsRed-N1 plasmids and TRAF1 and EGFP target genes, were linked with T4 DNA ligase, and IKK- alpha -pEGFP, TRAF1-pDsRed and recombinant plasmid were obtained. Then the joint products were transformed into alpha receptive bacteria to select positive clon.
2. identification of positive clones: screening positive clones with colony PCR and using the restriction endonuclease double enzyme to identify the positive clones. The results showed that three kinds of recombinant bodies were able to obtain the target fragments of 2.2Kb, 1.3Kb and 720bp by enzyme cut, and the size of the linked target genes. Further DNA sequencing analysis showed that all three recombinant bodies contained IKK- a, TRAF each. The coding regions of 1 and EGFP showed no mutation and frameshift, suggesting that IKK-, -pEGFP, TRAF1-pDsRed and EGFP-pDsRed three recombinants have been successfully constructed.
Two. Expression and identification of three recombinants of IKK- alpha -pEGFP, TRAF1-pDsRed and EGFP-pDsRed in eukaryotic cells
The three recombinant proteins were transfected into COS7 cells respectively. It was found that the IKK- alpha -pEGFP fusion protein (green fluorescence) was dispersed in the cytoplasm, and the TRAF1-pDsRed fusion protein (red fluorescence) was localized in the cell subregion. The expression of EGFP-pDsRed fusion protein could present both green fluorescence and red fluorescence, mainly in the cytoplasm. It indicated that IKK- alpha -pEGFP, The three fusion proteins of TRAF1-pDsRed and EGFP-pDsRed were successfully expressed in eukaryotic cells.
In this experiment, three recombinant IKK- alpha -pEGFP, TRAF1-pDsRed, EGFP-pDsRed were successfully constructed and expressed in eukaryotic cells, and the intracellular localization of the two molecules of IKK- A and TRAF1 was confirmed. The construction of the basis for the further study of the interaction between the two molecules and the interaction between the two molecules and the intracellular segments of TM-TNF- alpha was constructed by FRET technology. It also provides positive reference for FRET detection.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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