K562细胞消减cDNA文库的构建及初步鉴定  

Construction and Preliminary Identification of Subtracted cDNA Library of Leukemia Cell Line K562

作　　者：宋艳斌[1] 马文丽[1] 冯春琼[1] 石嵘[1] 毛向明[1] 张宝[1] 郑文岭 

机构地区：[1]南方医科大学分子生物学研究所,广东广州510515 [2]广州军区流花桥医院肿瘤分子生物学研究所,广东广州510010

出　　处：《癌症》2005年第5期631-633,共3页Chinese Journal of Cancer

基　　金：国家自然科学基金资助项目(No.39880032);广州市重点科技攻关资助项目(No.99-Z-022-01)~~

摘　　要：背景与目的:消减杂交技术是一种寻找和克隆差异基因的方法。本研究目的是通过快速、高质量构建消减cDNA文库,筛选K562细胞中差异表达基因。方法:以用限制性显示(restriction display,RD)技术制备的K562细胞cDNA片段作为消减对象,以正常人淋巴细胞的Sau3AⅠ酶解cDNA片段对其进行消减。经过消减后的RD片段被分组扩增出来,并克隆于T载体中,挑选阳性克隆进行测序并进行初步分析。结果:构建的K562细胞消减cDNA文库,包含360个阳性克隆,插入片断大小范围在200～800bp之间,选取50个克隆进行测序分析,结果为来源于42个已知基因。结论:利用自行建立的消减杂交法,成功构建了特异性K562细胞消减cDNA文库,文库质量可靠,可用于进一步筛选及克隆K562细胞中差异表达基因。BACKGROUND & OBJECTIVE: Subtractive hybridization technology is a common method to screen and clone differentially expressed genes. This study was to construct subtracted cDNA library of leukemia cell line K562, and screen for differentially expressed genes. METHODS: cDNA fragments of K562 cells (tester), prepared by restriction display (RD), were subtracted with the Sau3A Ⅰ-digested cDNA fragments of normal lymphocytes (driver). The subtracted cDNA fragments were re-amplified, and cloned into pMD18-T vectors. Positive clones were selected by blue-white screening. The inserts in plasmid were amplified by polymerase chain reaction (PCR), and some of which were sequenced. RESULTS: The subtracted library contained 360 positive clones with cDNA fragments distributed mainly from 200 to 800 bp. The 50 randomly sequenced clones were derived from 42 known genes. CONCLUSION: Specific subtracted cDNA library of K562 cells was successfully constructed with reliable quality, and may be used to further screen and clone differentially expressed genes of K562 cells.

关 键 词：K562细胞 消减杂交 限制性显示 CDNA文库 

分 类 号：R73-3[医药卫生—肿瘤]