老年性白内障与正常晶体上皮细胞中差异表达基因的筛选  

Screening of differentially expressed genes in epithelial cells of age-related cataract and normal lens

作　　者：刘燕[1] 刘新平[2] 药立波[2] 周健[1] 王吉村[2] 柴玉波[2] 胡丹[1] 苏景宽[3] 惠延年[1] 

机构地区：[1]第四军医大学西京医院眼科,陕西西安710033 [2]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710033 [3]唐都医院医教部,陕西西安710038

出　　处：《第四军医大学学报》2003年第12期1066-1069,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金资助项目 (30 0 0 0 1 85)

摘　　要：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 。AIM: To screen the differentially expressed genes in epithelial cells of age related cataract and normal lens. METHODS: Improved subtractive hybridization was used to obtain the differential cDNA.Then the large fragments of the differential cDNA were amplified by PCR and cloned into plasmid vector. The false positive clones were removed by dot blotting hybridization and the true positive fragments were sequenced and compared with those genes in GenBank, the novel sequences were further analyzed by computer. RESULTS: Six hundred clones of the subtractive library were selected at random and identified by restriction enzyme digestion,400 fragments which were ≥750 bp were obtained. One hundred and fifty clones were obtained after removal of the false positive clones by reverse dot blotting. Among the 50 fragments which were sequenced, 30 were the fragments of known genes(including 20 genes whose function have been known and 10 genes whose sequences are known but function havent been known), 3 were novel sequences. One of the 3 novel sequences had 45% similarity with zinc finger protein in the amino sequence, so this gene might be related to cell growth regulation and transcription. CONCLUSION: The improved subtractive hybridization is a rapid and efficient method to obtain the differentially expressed genes, which will be of an important meaning for finding the rare and new genes and identifying their functions.

关 键 词：消减杂交法 园翳内障 晶体/细胞学 上皮细胞 差异表达基因 

分 类 号：R776.1[医药卫生—眼科] Q7[医药卫生—临床医学]