本文選題：HMGN2 + 亞細(xì)胞定位　； 參考：《四川大學(xué)》2005年博士論文

【摘要】：內(nèi)源性抗菌肽是天然免疫的重要介質(zhì)，是炎癥反應(yīng)起保護(hù)作用的重要分子基礎(chǔ)。尋找新的抗菌肽分子一直是科學(xué)家夢(mèng)寐以求的愿望。高遷移率組蛋白N2(High Mobility Group Chromosal protein N2，HMGN2)是脊椎動(dòng)物和非脊椎動(dòng)物的細(xì)胞核中普遍存在的非組蛋白，目前對(duì)其功能還不完全清楚。在本實(shí)驗(yàn)室從人LAK細(xì)胞和人宮頸黏液中分離純化尋找抗菌肽過(guò)程中，發(fā)現(xiàn)HMGN2具有較強(qiáng)的抗革蘭氏陰性菌的作用，為進(jìn)一步證實(shí)其參予機(jī)體天然免疫的可能性，及其可能機(jī)制，本文重點(diǎn)研究其在細(xì)胞內(nèi)的定位及利用其α螺旋結(jié)構(gòu)域較強(qiáng)的跨膜能力及腫瘤導(dǎo)向力，構(gòu)建重組免疫毒素，以期用于腫瘤的靶向治療。 本文分三個(gè)部分。 第一部分是從本室已構(gòu)建的pGEX-1λT-HMGN2大腸桿菌表達(dá)載體經(jīng)異丙基-β-D-硫代半乳糖苷(IPTG)誘導(dǎo)高效表達(dá)出GST-HMGN2融合蛋白，進(jìn)一步用GST親合層析柱分離純化得到較純的GST-HMGN2融合蛋白，免疫新西蘭兔，成功制備出較高效價(jià)的HMGN2特異多克隆抗體，初步用于THP-1細(xì)胞中HMGN2的亞細(xì)胞定位分析，發(fā)現(xiàn)在LPS刺激下，免疫細(xì)胞化學(xué)染色顯示HMGN2不僅分布于單核吞噬細(xì)胞核，還分布于細(xì)胞漿，并且ELISA法也顯示可釋放到細(xì)胞外。 第二部分為進(jìn)行HMGN2的亞細(xì)胞定位分析，提取人LAK細(xì)胞總RNA，設(shè)計(jì)合成相應(yīng)引物，應(yīng)用RT-PCR從其總RNA中擴(kuò)增HMGN2 cDNA，以pcDNA3.1-myc-his和pEGFP-N1為載體，成功構(gòu)建出HMGN2真核表達(dá)載體，轉(zhuǎn)染Hela細(xì)胞，，轉(zhuǎn)染效率達(dá)70％～80％，用抗六組氨酸臂的單克隆抗體經(jīng)免疫細(xì)胞化學(xué)染色顯示經(jīng)pcDNA3.1-myc-his-HMGN2轉(zhuǎn)染的Hela細(xì)胞除細(xì)胞核外，胞漿亦明顯著色，未轉(zhuǎn)染的Hela細(xì)胞無(wú)論細(xì)胞核還是細(xì)胞漿均顯陰性；用兔抗人HMGN2多克隆抗體進(jìn)行的免疫細(xì)胞化學(xué)染色顯示轉(zhuǎn)染的Hela細(xì)胞與用抗六組氨酸臂的單克隆抗體檢測(cè)結(jié)果相同，而未轉(zhuǎn)染的Hela細(xì)胞核內(nèi)和胞漿也均有
[Abstract]:Endogenous antimicrobial peptides are important mediators of innate immunity and important molecular basis for the protection of inflammatory reaction. The search for new antimicrobial peptide molecules has been the dream of scientists. High mobility group Chromosal protein N2MGN2) is a common nonhistone in the nuclei of vertebrates and non-vertebrates. In the course of isolation and purification of antimicrobial peptides from human Lak cells and human cervical mucus in our laboratory, we found that HMGN2 has a strong effect on anti-Gram-negative bacteria, in order to further confirm the possibility and possible mechanism of HMGN2 participating in innate immunity. In this paper, we focus on its localization in cells and its strong transmembrane ability and tumor targeting ability of its 偽 -helix domain, and construct recombinant immunotoxin to be used for tumor targeting therapy. This paper is divided into three parts. In the first part, GST-HMGN2 fusion protein was induced from pGEX-1 位 T-HMGN2 expression vector induced by isopropyl- 尾 -D- thiogalactoside IPTGG, and purified by GST affinity chromatography. New Zealand rabbits were immunized with HMGN2 specific polyclonal antibodies (HMGN2), which were used for subcellular localization of HMGN2 in THP-1 cells. Immunocytochemical staining showed that HMGN2 was not only distributed in mononuclear phagocytes, but also in mononuclear phagocytes. It was also distributed in cytoplasm, and Elisa showed that it could be released out of the cell. The second part is the subcellular localization analysis of HMGN2, extraction of human Lak cell total RNAs, design and synthesis of corresponding primers. HMGN2 cDNAwas amplified by RT-PCR from its total RNA. HMGN2 eukaryotic expression vector was successfully constructed using pcDNA3.1-myc-his and pEGFP-N1 as vectors. HMGN2 eukaryotic expression vector was transfected into Hela cells. The transfection efficiency was 70%. The cytoplasm of Hela cells transfected with pcDNA3.1-myc-his-HMGN2 was also significantly stained by immunocytochemical staining with monoclonal antibody against the six-hisine arm, in addition to the nucleus, the cytoplasm of Hela cells transfected with pcDNA3.1-myc-his-HMGN2 was also significantly stained. The immunocytochemical staining of rabbit anti-human HMGN2 polyclonal antibody showed that the transfected Hela cells had the same results as the monoclonal antibodies against HMGN2 arms. However, the untransfected Hela cells were also found in the nucleus and cytoplasm of Hela.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

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相關(guān)期刊論文 前10條

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本文編號(hào)：2009803