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人树突状细胞cDNA质粒文库的构建及其大规模随机测序体系的建立 |
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章卫平 曹雪涛 万 涛 朱学军 袁正隆 何 龙
摘 要 目的：建立人树突状细胞(DC)的cDNA文库，通过大规模随机测序克隆免疫新分子。方法：来源于正常人外周血的单核细胞，体外经GM-CSF和IL-4培养，扩增出高纯度的DC，抽提纯化mRNA，转录成cDNA，定向插入pSPORT2.0载体，建立人DC的cDNA质粒文库；用ABI377全自动测序仪对该文库进行随机测序，将得到的EST在Sun-E450服务器中与EMBL及Swissprot数据库进行同源性比较，筛选出代表未知基因EST，然后在GCG软件中进行分析，对重要的未知EST克隆其全长cDNA。结果：所扩增的DC经流式细胞仪分析高表达MHC-Ⅰ、MHC-Ⅱ、B7、CDla和CD83等表面标志，能强烈刺激同种异体T淋巴细胞的体外增殖反应；建立的DC cDNA文库92%以上克隆有平均1.6 kb大小的插入片段；从所测的12 000余条EST中筛选出代表未知基因的3 000余条，克隆到109条全长新基因，包括属于细胞因子、细胞因子受体、趋化因子和粘附分子等家族的免疫新分子，部分全长基因已在Genebank中登录。结论：成功建立了人DC的cDNA基因文库及其大规模随机测序体系，并克隆到免疫分子。
关键词 树突状细胞 cDNA文库 大规模测序 免疫分子 基因克隆
Establishment of large scale sequencing system for the cDNA library of
human dendritic cells
ZHANG Wei-Ping, CAO Xue-Tao, WAN Tao et al.
Department of Immunology, Second Military Medical University, Shanghai 200433
Abstract Objective： To construct cDNA plasmid library of human dendritic cells (DC) and establish large-scale sequencing system for new molecule identification. Methods:The peripheral blood monocytes from normal adults were cultured in the presence of GM-CSF and IL-4 to generate highly purified human DC.DC were characterized by FACS analysis and MLR, and subjected to RNA extraction and poly(A)+RNA purification. The cDNA library was constructed by inserting the transcripted cDNA from human DC into pSPORT2.0 vector and identified by restrictive cleavage. The random clones from human DC library were picked for large scale sequencing by ABI377 automated sequencers to create our database of expressed sequence tags (ESTs), which were analyzed and processed by bioinformatic tools. Results:Monocyte-derived DC express high levels of CDla, CD83, MHC-Ⅰ, MHC-Ⅱ, CD40 and B7-1, and stimulate the proliferation of allogeneic T cells potently. More than 92% random clones from DC cDNA library have inserts averaging 1.6 kb. Among 12 000 currently available ESTs, 4.3% was identified to be functionally immune-related, and 25% represente[1] [2] 下一页
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