稳定高效表达BRP 44的PC-3细胞系的建立  

Establishment of PC-3 Cell Line Expressing BRP44 Stably and Efficiently

作　　者：白巍[1] 李黔生[1] 吴刚[1] 彭亮[1] 靳风烁[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所,重庆400042

出　　处：《华南国防医学杂志》2007年第3期6-9,共4页Military Medical Journal of South China

基　　金：国家自然科学基金资助项目(30400446)

摘　　要：目的建立稳定高效表达BRP 44的PC-3细胞系。方法下载BRP 44的全长mRNA序列,用Oligo 6进行引物设计,在上下游引物的5’引入相应的HindⅢ、EcoR I限制性内切酶识别序列及保护碱基。提取LNCaP细胞的总RNA反转录成cDNA进行PCR扩增,产物连接到真核表达载体pcDNA 3．1／myc-His A中构建成重组体。重组载体经酶切和测序鉴定后,阳离子脂质体转染法转染PC-3细胞、G 418筛选、Northern杂交检测并挑选出表达最强的亚克隆。结果成功构建BRP 44的真核表达载体并获得高效稳定表达的BRP 44基因的PC-3细胞亚克隆。结论高效稳定表达BRP44的PC-3细胞亚克隆可用于下一步研究。Objective To establish a PC-3 cell line stably and highly expressing BRP 44. Methods mRNA sequence of BRP 44 was downloaded. A pair of primers containing the sites of given restrictive endonuclease at both the 5＇ ends were designed by Oligo 6 and synthesized. Reverse transcription PCR of the total RNA extracted from LNCaP cell line was performed. The products of PCR were cloned into a highly efficient eukaryotic expression vector pcDNA 3.1/ myc-His A. The recombinants were sequenced and identified by restrictive endonuclease digestion and then transfected into the PC-3 cell line by lipofectamine 2000. The stable transfectants were screened by G 418. Cell subclones were isolated by gradient dilution. The cell subclones highly expressing BRP 44 were identified by northern blotting. Results The eukaryotic expression vector stably and efficiently expressing BRP 44 were constructed and the cell subclones stably and efficiently expressing BRP 44 were established. Conclusion The cell subclones stably and efficiently expressing BRP 44 can be used for the further study of the effect of BRP 44 on prostate cancer cell biological behavior.

关 键 词：BRP44 PC-3细胞系 稳定转染 前列腺癌 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]