發(fā)布時(shí)間：2018-04-17 19:45

  本文選題：核因子-κB(NF-κB) + p65亞基��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2005年碩士論文

【摘要】：核因子-κB(nuclear factor-κB,NF-κB)是一種重要的核轉(zhuǎn)錄因子,可被多種刺激因素激活,并易位至細(xì)胞核,與相應(yīng)的靶基因結(jié)合,啟動(dòng)基因轉(zhuǎn)錄。NF-κB的靶基因包括編碼細(xì)胞因子、粘附分子、誘導(dǎo)型一氧化氮合酶等的基因,因此與多種炎性疾病、自身免疫性疾病和腫瘤的發(fā)生有關(guān)。NF-κB已成為了疾病治療的重要靶標(biāo)。 本實(shí)驗(yàn)室在化學(xué)性急性肺損傷研究研究中也發(fā)現(xiàn),全氟異丁烯吸入中毒導(dǎo)致的急性肺損傷,其中NF-κB也表現(xiàn)為過度表達(dá),表明NF-κB的異常激活與急性肺損傷的發(fā)生密切相關(guān)。 噬菌體展示技術(shù)是尋找小肽拮抗分子的最有效的方法之一,該技術(shù)已在蛋白質(zhì)結(jié)構(gòu)研究、抗原表位分析、人工抗體制備、細(xì)胞因子拮抗劑的研究和尋找細(xì)胞內(nèi)傳遞蛋白等研究中有廣泛的應(yīng)用,并在尋找導(dǎo)向小分子方面已取得了令人振奮的進(jìn)展。 因此本研究通過基因工程技術(shù)構(gòu)建重組表達(dá)的NF-κB p65的表達(dá)載體,并對(duì)表達(dá)產(chǎn)物進(jìn)行分離、純化。以表達(dá)的GST-NF-κB p65(GST-p65)融合蛋白為靶標(biāo),利用噬菌體十二隨機(jī)肽庫篩選NF-κB p65亞基結(jié)合肽,為尋找NF-κB特異性抑制劑奠定基礎(chǔ)。 根據(jù)人NF-κB p65亞基基因序列設(shè)計(jì)引物,以pEGFP/p65質(zhì)粒為模板PCR擴(kuò)增得到NF-κB p65基因,將該基因克隆到克隆載體pGEM-T,獲得克隆重組質(zhì)粒pGEM-T/p65。將克隆重組質(zhì)粒中的目的基因片段進(jìn)行序列測(cè)定并與人NF-κB p65基因進(jìn)行比較,結(jié)果序列一致讀碼框沒有發(fā)生改變。從克隆重組質(zhì)粒上切下目的基因片段,將該基因克隆到谷胱甘肽S轉(zhuǎn)移酶(glutathione S-transferase,GST)融合表達(dá)載體pGEX-4T-2上,在大腸桿菌BL21中經(jīng)IPTG誘導(dǎo)表達(dá)。得到的GST-p65
[Abstract]:Nuclear factor-魏 B(nuclear factor- 魏 B (NF- 魏 B) is an important nuclear transcription factor, which can be activated by many stimuli and translocated to the nucleus, which binds to the corresponding target gene. The target gene that activates the gene transcription. NF- 魏 B includes encoded cytokines and adhesion molecules.Inducible nitric oxide synthase (iNOS) and other genes are involved in many inflammatory diseases, autoimmune diseases and tumorigenesis. NF- 魏 B has become an important target of disease treatment.In our laboratory, we also found that the acute lung injury induced by perfluoroisobutene inhalation was also overexpression of NF- 魏 B, which indicated that the abnormal activation of NF- 魏 B was closely related to the occurrence of acute lung injury.Phage display is one of the most effective methods to search for small peptide antagonist molecules. It has been used in protein structure analysis, antigen epitope analysis and preparation of artificial antibodies.The study of cytokine antagonists and the search for intracellular transfer proteins have been widely used, and have made exciting progress in the search for targeted small molecules.Therefore, the recombinant expression vector of NF- 魏 B p65 was constructed by genetic engineering, and the expression product was isolated and purified.The fusion protein of GST-NF- 魏 B p65 (GST-p65) was used to screen NF- 魏 B p65 subunit binding peptide using phage 12 random peptide library, which laid the foundation for searching for NF- 魏 B specific inhibitor.According to the sequence of human NF- 魏 B p65 subunit gene, the NF- 魏 B p65 gene was amplified by PCR using pEGFP/p65 plasmid as template. The gene was cloned into the clone vector pGEM-T, and the cloned recombinant plasmid pGEM-T / p65 was obtained.The target gene fragment in the cloned recombinant plasmid was sequenced and compared with the human NF- 魏 B p65 gene.The target gene fragment was cut off from the cloned recombinant plasmid and cloned into the fusion expression vector pGEX-4T-2 of glutathione S-transferase (GST), which was induced by IPTG in Escherichia coli BL21.Obtained GST-p65
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：Q789

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前3條

1 朱彥琪;動(dòng)脈內(nèi)皮損傷后炎癥反應(yīng)機(jī)制及L-精氨酸和自身骨髓單個(gè)核細(xì)胞干預(yù)效果的研究[D];復(fù)旦大學(xué);2005年

2 李紅良;鋅指蛋白A20對(duì)氧化性低密度脂蛋白介導(dǎo)的巨噬細(xì)胞與血管平滑肌細(xì)胞增殖和凋亡的影響及其分子機(jī)制的研究[D];中國協(xié)和醫(yī)科大學(xué);2005年

3 周媛;星形膠質(zhì)細(xì)胞在炎癥相關(guān)的神經(jīng)變性疾病發(fā)病機(jī)制中的作用研究[D];蘇州大學(xué);2006年

相關(guān)碩士學(xué)位論文 前3條

1 王偉;核因子κB在子宮內(nèi)膜異位癥發(fā)病機(jī)制中作用的研究[D];山西醫(yī)科大學(xué);2006年

2 金勇;胰島素抵抗大鼠動(dòng)脈血管內(nèi)皮炎癥與脂聯(lián)素關(guān)系的研究[D];中國醫(yī)科大學(xué);2007年

3 李澤信;腹部火器傷腸管穿透后肝內(nèi)NF-κB活性變化及其在肝損傷中的作用研究[D];石河子大學(xué);2007年

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本文編號(hào)：1765013