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
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靳西凤, 冉志华, 陈 翔, 萧树东
酵母细胞;SMART技术;酵母双杂交cDNA文库;同源重组,靳西凤,冉志华,陈翔,萧树东,通讯作者:,Constructionandcharacterizationofyeasttwo-hybridcDNAlibr
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靳西凤, 冉志华, 陈翔, 萧树东, 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
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
通讯作者: 冉志华, 200001, 上海市, 上海交通大学医学院附属仁济医院消化内科, 上海市消化疾病研究所. violetmoodee1210@126.com
电话: 021-63260930
收稿日期: 2006-10-31 接受日期: 2006-11-28
Construction and characterization of yeast two-hybrid cDNA library of hydroxycamptothecin-resistant human colon cancer cell line SW1116/HCPT in yeast cells
Xi-Feng Jin, Zhi-Hua Ran, Xiang Chen, Shu-Dong Xiao
Xi-Feng Jin, Zhi-Hua Ran, Xiang Chen, Shu-Dong Xiao,Renji Hospital, School of Medicine, Shanghai Jiao Tong University; Shanghai Institute of Digestive Diseases, Shanghai 200001, China
Supported byNational Natural Science Foundation of China, No. 30670942, and the Key Laboratory of Shanghai Municipal Commission of Science and Technology, No. 06DZ22027
Correspondence to:Zhi-Hua Ran, Renji Hospital, School of Medicine, Shanghai Jiao Tong University; Shanghai Institute of Digestive Diseases, Shanghai 20001, China. violetmoodee1210@126.com
Received:2006-10-31 Accepted:2006-11-28
Abstract
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)×106 recombinants were obtained from the cDNA library, and the recombination rate was 93 ......
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