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目的 :克隆胸腺细胞发育相关新基因。方法 :应用抑制性差减杂交技术 (suppressionsubtractivehybridiza tion ,SSH) ,构建了胸腺基质细胞系cDNA差减杂交文库 ,结合RACE及RT PCR进一步克隆并验证新基因的cD NA全长序列。结果 :筛选两个不同胸腺基质细胞系的差异表达基因 ,获得了差异表达的cDNA片段 ,克隆后挑选阳性克隆进行测序 ,成功克隆 8个新基因片段。对其中两个新基因克隆C5 5和C91进行Northern杂交分析 ,mR NA转录体长度分别为 1.4kb及 1.5kb ,C5 5的表达在两种胸腺基质细胞系中存在显著差异。进一步克隆了C5 5和C91两个新基因的cDNA全长序列 ,已被GenBank所接受。结论 :抑制性差减杂交技术是克隆新基因片段的有效方法 ;成功克隆 8个新基因片段及两个新基因的cDNA全长序列
Objective: To clone new genes related to thymocyte development. Methods: Suppression subtractive hybridization (SSH) was used to construct cDNA subtractive hybridization library of thymus stromal cell line. The full-length cD NA of the new gene was cloned and verified by RACE and RT PCR. Results: The differentially expressed genes of two different thymic stromal cell lines were screened and differentially expressed cDNA fragments were obtained. After cloning, positive clones were selected and sequenced. Eight new gene fragments were successfully cloned. Two new gene clones, C5 5 and C91, were analyzed by Northern blot. The length of mR NA transcripts were 1.4 kb and 1.5 kb, respectively. The expression of C5 5 was significantly different between the two thymic stromal cell lines. Further cloning of the full-length cDNA sequence of two novel genes, C5 5 and C91, has been accepted by GenBank. Conclusion: Suppression subtractive hybridization is an effective method for cloning new gene fragments. The full-length cDNA sequence of 8 new genes and two new genes were successfully cloned