人DC-SIGN蛋白克隆及免疫学功能初探

发布时间：2018-01-09 20:17

本文关键词：人DC-SIGN蛋白克隆及免疫学功能初探　出处：《重庆医科大学》2009年硕士论文　论文类型：学位论文

更多相关文章： DC-SIGN 转染 COS7细胞 免疫荧光

【摘要】： DC-SIGN全称是树突状细胞特异性细胞间粘附分子-3-结合非整合素分子(dendritic cell-specific intercellular adhesion molecule-3- grabbing nonintergrin,DC-SIGN),又称CD209,表达在未成熟的树突状细胞(immature dendritic cells,iDCs)表面,主要作用是捕获抗原和参与细胞间粘附。多种病原体通过自身不同结构成分与DC-SIGN结合,从而进入树突状细胞(dendritic cells,DCs),包括HIV、BCG、HCV、CMV、埃博拉病毒、利什曼原虫等[1-4]。其中,结核分枝杆菌(Mycobacterium tuberculosis,Mtb)通过其胞壁成分—带甘露糖帽的脂阿拉伯甘露聚糖(mannose-capped lipoarabinomannan, ManLAM)与DC-SIGN分子进行强有力结合,介导Mtb黏附、感染DCs,并经信号内传和交流,抑制iDCs成熟。Mtb隐藏在DCs中,通过某些机制,形成非溶酶体酸性隔室作为寄生屏障,从而逃避免疫监视和药物攻击,潜伏下来,成为日后结核复发的因素[4、5]。本研究首先预备以C57BL/6J小鼠为动物模型进行小鼠DC-SIGN研究。对小鼠DCs体外培养和流式细胞仪分析后,我们发现随着培养时间的推移,流式细胞仪所检测到的小鼠CD209表达量逐渐减少,在培养的第3天,流式细胞仪检测CD209的表达量极小,可以认为是0。在2007年之前的文献中,国外研究人员发现小鼠DC-SIGN相关蛋白组成复杂,具有多个同系物,在功能和结构上同人差异很大,从而指出小鼠作为模型研究DC-SIGN的价值还须进一步深入探讨[6-8]。经过讨论和专家指导,课题组将实验对象改为人的DCs。从人外周血分离DCs,培养后提取总RNA,RT-PCR得到编码DC-SIGN蛋白的基因片段,总长度1240bp,同表达绿色荧光的质粒pEGFP-C1重组,获得总长度为5938bp的重组质粒(命名为:DS-pEGFP-C1)。Lipofectamine2000脂质体转染COS7细胞,表达DC-SIGN绿色荧光融合蛋白(命名为:DS-EGFP)。Real-Time PCR定量工具细胞中mRNA的转录量为4.52×1011copies/ml。标记物绿色荧光蛋白连接在DC-SIGN胞内氨基端,在假设不影响其胞外碳端的抗原结合功能的情况下,我们最后用免疫荧光法进行了DS-EGFP与BCG结合的初步鉴定。研究结果:用RT-PCR扩增得到正确的目的基因片段并构建成真核表达载体DS-pEGFP-C1,转染COS7细胞,荧光定量PCR检测DS-pEGFP-C1转录mRNA拷贝量是4.52×1011copies/ml,激光扫描共焦显微镜检测证实DS-EGFP在COS7细胞表面表达,同时具有摄取BCG的功能。研究结论:本研究成功构建人DC-SIGN绿色荧光融合蛋白的真核表达载体DS-pEGFP-C1,表达了融合蛋白DS-EGFP。标记物连接在DC-SIGN蛋白氨基端,不影响DC-SIGN结合抗原。重组质粒可用于进一步筛选DC-SIGN基因序列中最佳RNAi干扰片段,为有效的RNAi打下基础。
[Abstract]:DC-SIGN is a dendritic cell-specific intercellular adhesion molecule-3-binding non-integrin molecule. Dendritic cell-specific intercellular adhesion molecule-3- grabbing. Nonintergrin. DC-SIGN, also called CD209, is expressed on the surface of immature dendritic cells, immature dendritic cells / iDCs. The main function is to capture antigens and participate in intercellular adhesion. Many pathogens bind to DC-SIGN through their own different structural components, thus entering dendritic cells into dendritic cells. DCsN, including HIV BCGV / CMV, Ebola virus, Leishmania protozoa, etc. [1-4] .Mycobacterium tuberculosis. The cell wall component of Mtb was mannose-capped lipoarabinomannan with mannose-capped capped arabinan with mannose cap. ManLAM) strongly binds to DC-SIGN molecules, mediates Mtb adhesion, infects DCS, and inhibits iDCs maturation. MTB is hidden in DCs through signal transmission and communication. Through some mechanisms, a non-lysosomal acidic compartment is formed as a parasitic barrier, thus avoiding immune surveillance and drug attacks, lurking down, and becoming a factor for the recurrence of tuberculosis in the future. [4,5]. In this study, C57BL / 6J mice were used as the animal model to study the DC-SIGN of mice. The mouse DCs was cultured in vitro and analyzed by flow cytometry. We found that with the passage of culture time, the expression of CD209 in mice detected by flow cytometry gradually decreased. On the third day of culture, the expression of CD209 detected by flow cytometry was very small. In the literature before 2007, foreign researchers found that mouse DC-SIGN related proteins have complex composition, have multiple homologues, and differ greatly in function and structure. It is pointed out that the value of studying DC-SIGN in mice as a model should be further explored. [After discussion and expert guidance, our group changed the experimental object into human DCs.isolate DCS from human peripheral blood and extract total RNA after culture. The gene fragment encoding DC-SIGN protein was obtained by RT-PCR. The total length of the gene was 1240bp. the plasmid pEGFP-C1 was recombined with the expression of green fluorescence. A total length of 5938 BP recombinant plasmid was obtained and transfected into COS7 cells with liposome: 1. DS-pEGFP-C1. Lipofectamine2000. Expression of DC-SIGN green fluorescent fusion protein. Real-Time. The transcription amount of mRNA in PCR quantitative tool cells was 4.52 脳 10 11 copes / ml. The green fluorescent protein (GFP) was linked to the amino terminal of DC-SIGN. Under the assumption that the antigen-binding function of the extracellular carbon terminal was not affected, we finally identified the binding of DS-EGFP to BCG by immunofluorescence method. Results: the correct target gene fragment was obtained by RT-PCR amplification and the eukaryotic expression vector DS-pEGFP-C1 was constructed and transfected into COS7 cells. The copy amount of DS-pEGFP-C1 transcribed mRNA detected by fluorescence quantitative PCR was 4.52 脳 1011copes / ml. Laser scanning confocal microscopy confirmed that DS-EGFP was expressed on the surface of COS7 cells and had the function of uptake of BCG. Conclusion: in this study, the eukaryotic expression vector DS-pEGFP-C1 of human DC-SIGN green fluorescent fusion protein was successfully constructed. The fusion protein DS-EGFP. was linked to the amino terminal of DC-SIGN protein. The recombinant plasmid can be used to further screen the best RNAi interference fragment in the DC-SIGN gene sequence and lay the foundation for effective RNAi.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.111

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