PTD-OD-HA融合蛋白对Bcr-Abl阳性细胞促凋亡抑增殖效应的研究
发布时间：2018-08-13 19:57
【摘要】：Bcr/Abl融合蛋白由多个功能域组成,其中位于Bcr N端1～72氨基酸内的OD域(oligomerization domain,OD)介导Bcr/Abl寡聚化,是Bcr/Abl自身磷酸化,导致分子构型改变,最终异常激活Abl内酪氨酸激酶的关键因素。因而我们推测通过体外合成OD蛋白转导入细胞干扰Bcr/Abl寡聚化,有望成为CML治疗的新策略。我们选择蛋白转导域(protein transduction domain,PTD)系统携带外源性OD蛋白进入细胞,同时加入HA标签以便后续研究的免疫学检测。 本实验通过构建、表达、纯化PTD-OD-HA融合蛋白,并将融合蛋白转导入CML细胞,检测细胞凋亡及增殖方面的效应,为后期开展动物体内研究奠定了基础。本课题采用的主要试验方法如下: 1.PTD-OD-HA融合蛋白的克隆构建、原核表达、纯化及鉴定:通过分步克隆,分别将OD、HA和PTD基因片段顺次克隆入pET32a(+)载体,经酶切与测序鉴定后,将序列正确的pPTD-OD-HA重组质粒转化大肠杆菌BL21(DE3)菌株,用IPTG诱导PTD-OD-HA融合蛋白表达,用His·Bind树脂纯化目的蛋白,纯化的蛋白用SDS-PAGE考马斯亮蓝染色和western blot进行鉴定。同时纯化对照蛋白OD-HA。 2. PTD-OD-HA融合蛋白对Bcr/Abl阳性细胞凋亡的影响:通过流式细胞术、DNA ladder实验及电镜检测细胞凋亡;RT-PCR和western blot检测凋亡相关基因bax、bcl-2 mRNA和蛋白水平的改变。 3. PTD-OD-HA融合蛋白对Bcr/Abl阳性细胞增殖的影响:通过MTT实验、流式细胞术、瑞氏染色以及电镜观察细胞周期和形态改变;RT-PCR和western blot检测周期相关基因cyclin D1、c-myc mRNA和蛋白水平的改变。 通过以上实验,得到以下主要的结果或结论: 1.成功地构建了pPTD-OD-HA和OD-HA原核表达载体,成功地表达出PTD-OD-HA和OD-HA融合蛋白,其最佳诱导表达条件分别是: 0.1mmol/L IPTG 37℃诱导6h,1.0mmol/L IPTG 37℃诱导4h;经SDS-PAGE考马斯亮蓝染色和western blot验证了纯化蛋白的正确性。 2. PTD-OD-HA融合蛋白进入细胞后,能特异性地引发Bcr/Abl阳性细胞发生凋亡,对Bcr/Abl阴性细胞的凋亡不产生影响;DNA ladder实验检测到凋亡细胞因DNA断裂而产生的典型的梯状条带;电镜观察到凋亡小体;RT-PCR及western blot检测凋亡相关基因bax mRNA和蛋白水平都升高,而bcl-2 mRNA和蛋白水平都降低。 3. PTD-OD-HA蛋白作用于K562和BaF3-P210两种Bcr/Abl阳性细胞后,MTT实验观察到PTD-OD-HA融合蛋白能减慢Bcr/Abl阳性细胞生长;流式细胞术检测发现细胞被阻滞在G1期;瑞氏染色以及电镜观察细胞形态发生改变,胞浆中出现大量的空泡,线粒体普遍肿胀,溶酶体增多;RT-PCR和western blot检测到周期相关基因cyclin D1、c-myc mRNA和蛋白水平均都下降了。
[Abstract]:The Bcr/Abl fusion protein is composed of several functional domains, among which the oligomerization domain (oligomerization domain OD), which is located in the N-terminal of Bcr, mediates Bcr/Abl oligomerization, which is the key factor of Bcr/Abl self-phosphorylation, leading to molecular configuration change and ultimately abnormal activation of tyrosine kinase in Abl. Therefore, we speculate that OD protein transduction into cells interferes with Bcr/Abl oligomerization in vitro, which may be a new strategy for CML therapy. We selected the protein transduction domain (protein transduction domain) system to carry exogenous OD protein into the cells and add HA tag for further immunological detection. In this experiment, the PTD-OD-HA fusion protein was constructed, expressed and purified, and the fusion protein was transferred into CML cells to detect the effects of apoptosis and proliferation, which laid a foundation for the research in vivo. The main methods used in this study are as follows: cloning, prokaryotic expression, purification and identification of 1.PTD-OD-HA fusion protein: by step cloning, the ODHA and PTD gene fragments were cloned into pET32a () vector, respectively. After restriction endonuclease digestion and sequencing, the recombinant plasmid of pPTD-OD-HA was transformed into E. coli BL21 (DE3) strain. PTD-OD-HA fusion protein was induced by IPTG, the target protein was purified by His Bind resin, and the purified protein was identified by SDS-PAGE Coomassie brilliant blue staining and western blot. The control protein OD-HA.2 was purified simultaneously. The effect of PTD-OD-HA fusion protein on apoptosis of Bcr/Abl positive cells was analyzed by flow cytometry and electron microscopy. The changes of apoptosis related gene bcl-2 mRNA and protein were detected by RT-PCR and western blot. 3. The effect of PTD-OD-HA fusion protein on the proliferation of Bcr/Abl positive cells: the changes of cell cycle and morphology were observed by MTT assay, flow cytometry, Rayleigh staining and electron microscopy. The changes of cell cycle related gene cyclin D1 c-myc mRNA and protein were detected by western blot and reverse transcription-polymerase chain reaction (RT-PCR). Through the above experiments, the following main results or conclusions are obtained: 1. The prokaryotic expression vectors of pPTD-OD-HA and OD-HA were successfully constructed and the fusion proteins of PTD-OD-HA and OD-HA were successfully expressed. The optimal expression conditions were as follows: 1. 0 mmol / L IPTG was induced at 0.1mmol/L IPTG 37 鈩，
本文编号：2182020
[Abstract]:The Bcr/Abl fusion protein is composed of several functional domains, among which the oligomerization domain (oligomerization domain OD), which is located in the N-terminal of Bcr, mediates Bcr/Abl oligomerization, which is the key factor of Bcr/Abl self-phosphorylation, leading to molecular configuration change and ultimately abnormal activation of tyrosine kinase in Abl. Therefore, we speculate that OD protein transduction into cells interferes with Bcr/Abl oligomerization in vitro, which may be a new strategy for CML therapy. We selected the protein transduction domain (protein transduction domain) system to carry exogenous OD protein into the cells and add HA tag for further immunological detection. In this experiment, the PTD-OD-HA fusion protein was constructed, expressed and purified, and the fusion protein was transferred into CML cells to detect the effects of apoptosis and proliferation, which laid a foundation for the research in vivo. The main methods used in this study are as follows: cloning, prokaryotic expression, purification and identification of 1.PTD-OD-HA fusion protein: by step cloning, the ODHA and PTD gene fragments were cloned into pET32a () vector, respectively. After restriction endonuclease digestion and sequencing, the recombinant plasmid of pPTD-OD-HA was transformed into E. coli BL21 (DE3) strain. PTD-OD-HA fusion protein was induced by IPTG, the target protein was purified by His Bind resin, and the purified protein was identified by SDS-PAGE Coomassie brilliant blue staining and western blot. The control protein OD-HA.2 was purified simultaneously. The effect of PTD-OD-HA fusion protein on apoptosis of Bcr/Abl positive cells was analyzed by flow cytometry and electron microscopy. The changes of apoptosis related gene bcl-2 mRNA and protein were detected by RT-PCR and western blot. 3. The effect of PTD-OD-HA fusion protein on the proliferation of Bcr/Abl positive cells: the changes of cell cycle and morphology were observed by MTT assay, flow cytometry, Rayleigh staining and electron microscopy. The changes of cell cycle related gene cyclin D1 c-myc mRNA and protein were detected by western blot and reverse transcription-polymerase chain reaction (RT-PCR). Through the above experiments, the following main results or conclusions are obtained: 1. The prokaryotic expression vectors of pPTD-OD-HA and OD-HA were successfully constructed and the fusion proteins of PTD-OD-HA and OD-HA were successfully expressed. The optimal expression conditions were as follows: 1. 0 mmol / L IPTG was induced at 0.1mmol/L IPTG 37 鈩，
本文编号：2182020
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