利用抑制性减数杂交技术(SSH)研究IL-10抑制表达的新基因

Isolation of genes that are inhibited by IL- 10 in U937 cells by suppression subtractive hybridization (SSH)

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

Objective To isolate the genes that are inhibited by IL 10. Methods Prepare the U937 cells cultured with PHA and the U937 cells cultured with PHA and IL 10, extract the polyA RNA, synthesize the dscDNA by using reverse transcription PCR, get the differentially expressed genes by suppression subtractive hybridization(SSH). Results Fifteen genes whose expressions were probably inhibited by IL 10 were isolated by SSH. Six of them were novel genes revealed by GenBank searching. Northern blot showed that the expression of three novel genes could be inhibited by IL 10. The full length cDNA sequence of one gene has been obtained by EST assembling and represents a novel C C family chemokine. The gene′s accession number in GenBank is AF096985. Conclusion SSH is an effective method for isolating the differentially expressed genes, IL 10 can inhibit the expression of some genes including an unknown cytokine.