PTD-OD-HA融合蛋白對Bcr-Abl陽性細(xì)胞促凋亡抑增殖效應(yīng)的研究
發(fā)布時間：2018-08-13 19:57
【摘要】：Bcr/Abl融合蛋白由多個功能域組成,其中位于Bcr N端1～72氨基酸內(nèi)的OD域(oligomerization domain,OD)介導(dǎo)Bcr/Abl寡聚化,是Bcr/Abl自身磷酸化,導(dǎo)致分子構(gòu)型改變,最終異常激活A(yù)bl內(nèi)酪氨酸激酶的關(guān)鍵因素。因而我們推測通過體外合成OD蛋白轉(zhuǎn)導(dǎo)入細(xì)胞干擾Bcr/Abl寡聚化,有望成為CML治療的新策略。我們選擇蛋白轉(zhuǎn)導(dǎo)域(protein transduction domain,PTD)系統(tǒng)攜帶外源性O(shè)D蛋白進(jìn)入細(xì)胞,同時加入HA標(biāo)簽以便后續(xù)研究的免疫學(xué)檢測。 本實驗通過構(gòu)建、表達(dá)、純化PTD-OD-HA融合蛋白,并將融合蛋白轉(zhuǎn)導(dǎo)入CML細(xì)胞,檢測細(xì)胞凋亡及增殖方面的效應(yīng),為后期開展動物體內(nèi)研究奠定了基礎(chǔ)。本課題采用的主要試驗方法如下: 1.PTD-OD-HA融合蛋白的克隆構(gòu)建、原核表達(dá)、純化及鑒定:通過分步克隆,分別將OD、HA和PTD基因片段順次克隆入pET32a(+)載體,經(jīng)酶切與測序鑒定后,將序列正確的pPTD-OD-HA重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21(DE3)菌株,用IPTG誘導(dǎo)PTD-OD-HA融合蛋白表達(dá),用His·Bind樹脂純化目的蛋白,純化的蛋白用SDS-PAGE考馬斯亮藍(lán)染色和western blot進(jìn)行鑒定。同時純化對照蛋白OD-HA。 2. PTD-OD-HA融合蛋白對Bcr/Abl陽性細(xì)胞凋亡的影響:通過流式細(xì)胞術(shù)、DNA ladder實驗及電鏡檢測細(xì)胞凋亡;RT-PCR和western blot檢測凋亡相關(guān)基因bax、bcl-2 mRNA和蛋白水平的改變。 3. PTD-OD-HA融合蛋白對Bcr/Abl陽性細(xì)胞增殖的影響:通過MTT實驗、流式細(xì)胞術(shù)、瑞氏染色以及電鏡觀察細(xì)胞周期和形態(tài)改變;RT-PCR和western blot檢測周期相關(guān)基因cyclin D1、c-myc mRNA和蛋白水平的改變。 通過以上實驗,得到以下主要的結(jié)果或結(jié)論: 1.成功地構(gòu)建了pPTD-OD-HA和OD-HA原核表達(dá)載體,成功地表達(dá)出PTD-OD-HA和OD-HA融合蛋白,其最佳誘導(dǎo)表達(dá)條件分別是: 0.1mmol/L IPTG 37℃誘導(dǎo)6h,1.0mmol/L IPTG 37℃誘導(dǎo)4h;經(jīng)SDS-PAGE考馬斯亮藍(lán)染色和western blot驗證了純化蛋白的正確性。 2. PTD-OD-HA融合蛋白進(jìn)入細(xì)胞后,能特異性地引發(fā)Bcr/Abl陽性細(xì)胞發(fā)生凋亡,對Bcr/Abl陰性細(xì)胞的凋亡不產(chǎn)生影響;DNA ladder實驗檢測到凋亡細(xì)胞因DNA斷裂而產(chǎn)生的典型的梯狀條帶;電鏡觀察到凋亡小體;RT-PCR及western blot檢測凋亡相關(guān)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-P210兩種Bcr/Abl陽性細(xì)胞后,MTT實驗觀察到PTD-OD-HA融合蛋白能減慢Bcr/Abl陽性細(xì)胞生長;流式細(xì)胞術(shù)檢測發(fā)現(xiàn)細(xì)胞被阻滯在G1期;瑞氏染色以及電鏡觀察細(xì)胞形態(tài)發(fā)生改變,胞漿中出現(xiàn)大量的空泡,線粒體普遍腫脹,溶酶體增多;RT-PCR和western blot檢測到周期相關(guān)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，
本文編號：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，
本文編號：2182020
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