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目的 :筛选老年性白内障与正常晶体上皮细胞的差异表达基因 .方法 :利用改良消减杂交法获得差异cDNA ,利用PCR法扩增其中大片段差异cDNA ,将其克隆入质粒载体 ,通过反向点杂交排除假阳性克隆后 ,阳性克隆测序与Gen Bank中的序列进行比较 ,新序列进行计算机分析 .结果 :在文库中随机挑选约 6 0 0个克隆酶切鉴定 ,获得≥ 75 0bp片段4 0 0个 .经反向点杂交 ,排除假阳性克隆后 ,共得到阳性克隆15 0个 ;测序的 5 0个片段中 ,有 30个已知基因 (包括 2 0个功能已知基因、10个序列已知但功能未知基因 ) ,3个新序列 .新序列中有一个的氨基酸序列与假想的锌指蛋白有 4 5 %同源性 ,可能参与细胞生长调控与代谢 .结论 :改良消减杂交法可以快速有效地获得老年性白内障差异表达基因 ,对稀有基因及新基因的发现与功能鉴定有重要意义
OBJECTIVE: To screen differentially expressed genes in senile cataract and normal lens epithelial cells.Methods: The differential cDNAs were obtained by improved subtractive hybridization, and the large cDNA fragments were amplified by PCR and cloned into plasmid vector. After excluding the false positive clones, the positive clones were sequenced and compared with those in Gen Bank, and the new sequences were analyzed by computer.Results: About 600 clones were randomly selected in the library to identify 400 clones with ≥ 75 0bp fragments A total of 150 positive clones were obtained by reverse dot blot hybridization. Among the 50 fragments sequenced, 30 were known genes (including 20 known genes and 10 were known But unknown function gene), three new sequences.The amino acid sequence of the new sequence has 45% homology with the hypothetical zinc finger protein, which may be involved in the regulation of cell growth and metabolism.Conclusion: The improved subtractive hybridization method can be rapid and effective To obtain differentially expressed genes in senile cataracts, which is of great significance for the discovery and functional identification of rare genes and new genes