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目的 利用抑制性减数杂交 (SSH)技术筛选IL 10抑制表达的新基因。方法 以PHA刺激的U937细胞为tester,以PHA刺激同时IL 10抑制的U937细胞为driver,提取mRNA ,反转录构建cDNA文库 ,利用SSH技术筛选IL 10抑制表达的新基因。结果 通过SSH技术 ,发现了 15个可能的IL 10抑制表达的基因 ,其中 6个与GenBank中已公布的表达序列检签 (expressedsequencetag ,EST)片段没有明显同源性 ,为新基因 ,其中之一经EST拼接 ,得到全长cDNA ,为一种新的C C家族趋化因子 ,已被GenBank收录 ,收录号码为AF0 96 985。挑选 4个新基因进行Northernblot分析 ,发现IL 10可抑制其中 3个新基因表达。结论 SSH技术是一种高效的差异基因筛选方法 ,IL 10可抑制多种包括新细胞因子基因的表达
OBJECTIVE: To screen novel genes that inhibit the expression of IL-10 by inhibition subtractive hybridization (SSH). Methods U937 cells stimulated by PHA and driver of U937 cells stimulated by PHA were treated by PHA. The mRNA was extracted and reverse transcribed into cDNA library. The new gene of IL - 10 was screened by SSH. Results Fifteen possible genes that were inhibited by IL-10 were found by SSH. Six of them showed no significant homology with published sequence tags (ESTs) published in GenBank, and one of them EST splicing to obtain a full-length cDNA, a new CC family of chemokines, has been included in GenBank accession number AF0 96 985. Four new genes were selected for Northern blot analysis and IL-10 was found to inhibit the expression of three new genes. Conclusion SSH is a highly efficient method for screening differential genes and IL-10 inhibits the expression of a variety of genes, including novel cytokines