Cbl-b shRNA稳定转染乳腺癌MDA-MB-231细胞系的构建  

Construction of breast cancer MDA-MB-231 cell line transfected by Cbl-b shRNA

作　　者：张凌云[1] 石晶[1] 滕月娥[1] 张晔[1] 曲秀娟[1] 刘云鹏[1] 

机构地区：[1]中国医科大学附属第一医院,沈阳110001

出　　处：《山东医药》2011年第52期31-33,共3页Shandong Medical Journal

基　　金：国家青年科学基金资助项目(30700807);辽宁省教育厅重点实验室项目(2008S246)

摘　　要：目的构建靶向Cbl-b基因的短发夹环RNA(shRNA)真核质粒表达载体,建立Cbl-b shRNA稳定转染人乳腺癌MDA-MB-231细胞系,为探讨Cbl-b的生物学功能奠定基础。方法设计Cbl-b shRNA序列BLAST进行同源性分析。化学合成法合成shRNA序列,将配对的单链合成双链,以T4连接酶连接于pSilencerTM 4.1-CMV表达载体,加入感受态细胞。将菌液涂布于含有氨苄青霉素的LB平板,筛选阳性菌落,测序确认后进行大量复制、扩增,提取重组质粒。脂质体法将上述质粒转染至MDA-MB-231细胞中,G418持续压力选择和有限稀释法获得稳定转染的细胞系。结果 DNA测序证实靶向Cbl-b基因的shRNA真核质粒表达载体构建正确,G418压力筛选出阳性克隆,Western blot检测发现MDA-MB-231/Cbl-b shRNA转染细胞株中Cbl-b蛋白表达水平明显下调。结论成功构建了基因沉默Cbl-b的MDA-MB-231细胞株,为进一步探讨其生物学功能奠定实验基础。Objective To construct the targeting Cbl-b gene short hairpin RNA（shRNA） eukaryotic plasmid expression vector,and to obtain the stable transfected MDA-MB-231 cell lines.Methods Cbl-b shRNA sequence and the control target sequences were chemically synthesized and inserted into pRNA-U6.1/Neo vector after annealing,which were confirmed by sequencing.Then the recombinant plasmids were transfected into MDA-MB-231 cells with lipofectin and the expression of Cbl-b was detected by Western blot.Results Cbl-b shRNA,and blank control template was sucessfully built on the pRNA-U6.1/Neo vector.Then the recombinant plasmids were successfully infected in MDA-MB-231 cells,and Cbl-b protein expression was down-regulated in MDA-MB-231/Cbl-b shRNA cells.Conclusion MDA-MB-231/Cbl-b shRNA breast cancer cells can be used for the study on the biological function of Cbl-b.

关 键 词：乳腺肿瘤 CBL-B 短发夹环RNA MDA-MB-231 转染 

分 类 号：R73-3[医药卫生—肿瘤]