结肠癌羟基喜树碱耐药细胞SW1116/HCPT酵母双杂交cDNA文库的构建和鉴定  

Construction and characterization of yeast two-hybrid cDNA library of hydroxycamptothecin-resistant human colon cancer cell line SW1116/HCPT in yeast cells

作　　者：靳西凤[1] 冉志华[1] 陈翔[1] 萧树东[1] 

机构地区：[1]上海交通大学医学院附属仁济医院,上海市消化疾病研究所,上海市200001

出　　处：《世界华人消化杂志》2007年第5期500-504,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金项目;No.30670942;上海市科委重点实验室项目;No.06DZ22027~~

摘　　要：目的:构建羟基喜树碱耐药结肠癌细胞株SW1116/HCPT细胞的酵母双杂交cDNA文库.方法:用TRIzol从SW1116/HCPT细胞提取总RNA,采用CLONTECH SMART(switching mechanism at 5' end of RNA transcript)技术和同源重组(homologous recombination)的方法构建SW1116/HCPT细胞的cDNA文库.结果:提取的RNA的A260/A280为1.98,甲醛变性琼脂糖凝胶电泳示出28SrRNA、18SrRNA及5SrRNA3条特异性带,28S与18S带浓度及亮度比值约为2.文库dscDNAsmear较长分布在0.1-5kb,成长瀑布条带,而且在接近1kb的区域有密集的带子,为高丰度表达基因.依据生长菌落计数,共获得(1.2-1.9)×106个转化子,重组率达93.7%,文库插入片段大小为0.2-5kb.结论:成功构建了SW1116/HCPT cDNA文库.AIM： To construct a yeast two-hybrid cDNA library of hydroxycamptothecin-resistant human colon cancer cell line SW1116/HCPT in yeast cells. METHODS： The total RNA of SW1116/HCPT cells was extracted using TRIzol method. Clontech SMART （switching mechanism at 5＇end of RNA transcript） technique and homologous recombination mediated approach were used to construct cDNA library of SW1116/HCPT cells. RESULTS： The ratio of A260 to A280 value of the extracted RNA was 1.98. A total of 3 bands （28S rRNA, 18S rRNA and 5S rRNA） were exhibited by agar gel electrophoresis, and the ratio of 28S rRNA and 18S rRNA was about 2. The ds cDNA smear in cDNA library was between 0.1-5 kb in length, with high expression at the 1-kb region. A total of （1.2-1.9）×10^6 recombinants were obtained from the cDNA library, and the recombination rate was 93.7%. The amplified polymerase chain reaction （PCR） fragments were between 0.2 and 5 kb in length. CONCLUSION： The yeast two-hybrid cDNA li- brary of SW1116/HCPT is successfully constructed by Clontech SMART technique in yeast cell.

关 键 词：酵母细胞 SMART技术 酵母双杂交cDNA文库 同源重组 

分 类 号：R735.35[医药卫生—肿瘤]