HMGN2的亚细胞定位及其重组免疫毒素的研究

发布时间：2018-06-12 13:19

本文选题：HMGN2 + 亚细胞定位　；参考：《四川大学》2005年博士论文

【摘要】：内源性抗菌肽是天然免疫的重要介质，是炎症反应起保护作用的重要分子基础。寻找新的抗菌肽分子一直是科学家梦寐以求的愿望。高迁移率组蛋白N2(High Mobility Group Chromosal protein N2，HMGN2)是脊椎动物和非脊椎动物的细胞核中普遍存在的非组蛋白，目前对其功能还不完全清楚。在本实验室从人LAK细胞和人宫颈黏液中分离纯化寻找抗菌肽过程中，发现HMGN2具有较强的抗革兰氏阴性菌的作用，为进一步证实其参予机体天然免疫的可能性，及其可能机制，本文重点研究其在细胞内的定位及利用其α螺旋结构域较强的跨膜能力及肿瘤导向力，构建重组免疫毒素，以期用于肿瘤的靶向治疗。本文分三个部分。第一部分是从本室已构建的pGEX-1λT-HMGN2大肠杆菌表达载体经异丙基-β-D-硫代半乳糖苷(IPTG)诱导高效表达出GST-HMGN2融合蛋白，进一步用GST亲合层析柱分离纯化得到较纯的GST-HMGN2融合蛋白，免疫新西兰兔，成功制备出较高效价的HMGN2特异多克隆抗体，初步用于THP-1细胞中HMGN2的亚细胞定位分析，发现在LPS刺激下，免疫细胞化学染色显示HMGN2不仅分布于单核吞噬细胞核，还分布于细胞浆，并且ELISA法也显示可释放到细胞外。第二部分为进行HMGN2的亚细胞定位分析，提取人LAK细胞总RNA，设计合成相应引物，应用RT-PCR从其总RNA中扩增HMGN2 cDNA，以pcDNA3.1-myc-his和pEGFP-N1为载体，成功构建出HMGN2真核表达载体，转染Hela细胞，，转染效率达70％～80％，用抗六组氨酸臂的单克隆抗体经免疫细胞化学染色显示经pcDNA3.1-myc-his-HMGN2转染的Hela细胞除细胞核外，胞浆亦明显着色，未转染的Hela细胞无论细胞核还是细胞浆均显阴性；用兔抗人HMGN2多克隆抗体进行的免疫细胞化学染色显示转染的Hela细胞与用抗六组氨酸臂的单克隆抗体检测结果相同，而未转染的Hela细胞核内和胞浆也均有
[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 伪 -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.
【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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