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目的构建系统性红斑狼疮(systemiclupuserythematosus,SLE)外周血单个核细胞基因表达系列分析(serialanalysisofgeneexpression,SAGE)文库。方法从3例家族性SLE患者外周血单个核细胞分离PolyARNA,采用改良的SAGE技术构建SAGE文库,随机挑选阳性克隆进行菌落PCR鉴定及对部分阳性克隆进行测序。结果初步构建的SAGE文库已获得600个克隆,随机取50个进行菌落聚合酶链反应(PCR)分析,其中43个克隆中质粒插入片段大于300bp,占86%。部分克隆测序证实含有序列正确的SAGE标签。结论应用改良SAGE的技术成功构建了SLE外周血单个核细胞SAGE文库,为筛选SLE患者差异表达基因奠定了基础。
Objective To construct a serialanalysis of gene expression (SAGE) library of systemic lupus erythematosus (SLE). Methods PolyARNA was isolated from peripheral blood mononuclear cells from 3 patients with familial SLE. SAGE library was constructed by modified SAGE technique. Positive clones were randomly selected for colony PCR identification and partial positive clones were sequenced. Results A total of 600 clones were obtained from the SAGE library. Fifty randomly selected colonies were subjected to colony polymerase chain reaction (PCR) analysis. Of the 43 clones, plasmid inserts were over 300 bp, accounting for 86%. Partial cloning sequencing confirmed that the sequence contains the correct SAGE tag. Conclusion The SAGE library of SLE peripheral blood mononuclear cells has been successfully constructed using the improved SAGE technique, which lays the foundation for the screening of differentially expressed genes in patients with SLE.