人BMI-1基因真核表达载体构建及其在人乳腺癌细胞系MDA-MB-468细胞中的表达  

Construction of human bmi-1 eukaryotic expression vector and its expression in mammary cancer cell line MDA-MB-468

作　　者：王影利[1,2] 邓青[1,2] 尹玉慧[1,2] 李惠翔[1,2] 

机构地区：[1]郑州大学基础医学院,河南郑州450052 [2]郑州大学第一附属医院,河南郑州450052

出　　处：《中医临床研究》2010年第9期98-101,103,共5页Clinical Journal Of Chinese Medicine

摘　　要：目的：构建人Bmi-1基因的真核表达载体pcDNA3．1（＋）-Bmi-1，转染人乳腺癌细胞株MDA-MB-468细胞中并检测其表达。方法：RT-PCR检测Bmi．1mRNA在乳腺癌细胞系MCF-7及MDA-MB-468细胞中的表达水平；从MCF-7细胞中提取总RNA，逆转录为cDNA，以cDNA为模板，扩增Bmi-1基因序列，将含有Bmi-1全长编码序列的PCR产物经TA克隆后经PCR、酶切验证，亚克隆入真核表达载体pcDNA3．1（＋）中，构建重组质粒pcDNA3．1（＋）-Bmi-1，转化至大肠杆菌后，酶切、测序鉴定；将重组质粒转染到MDA-MB-468中，48h后RT-PCR检测转染前后Bmi-1mRNA及hTERTmRNA的表达变化。结果：Bmi-1mRNA在MCF-7细胞中表达较高，而在MDA-MB-468细胞中仅适量表达。成功从MCF-7细胞中克隆获得Bmi-1基因并构建真核表达载体。转染后在MDA-MB-468中检测到Bmi-1mRNA表达上调，其过表达上调hTERTmRNA的表达。结论：成功克隆和建立人Bmi-1基因真核表达载体；pcDNA3．1（＋）-Bmi-1能在MDA-MB-468中表达；为进一步研究Bmi-1基因在细胞中的功能奠定了基础。Objective： To construct human Bmi-1 gene eukaryotic expression vector which was transfected into mammary carcinoma cell line MDA-MB-468 and its expression level was measured. Methods： The expression of Bmi-1 mRNA was determined by RT-PCR in human breast cancer cell lines MCF-7 and MDA-MB-468. The total RNA was extracted from MCF-7 cell line,which was used to reverse transcript for cDNA. The cDNA was used to amplify the Bmi-1 gene. The PCR product containing the entire coding sequence of Bmi-1 was cloned to a T vector and the recombinant vector was tested by PCR and restriction enzyme digestion after TA clone. Then the PCR product of Bmi-1 was subcloned to the eukaryotic expression vector pcDNA3.1（ ＋ ）. The recombinant eukaryotic expression vector was transformed to E. coli, measured by restriction enzyme digestion and sequence analysis. The Eukaryotic Expression Vector pcDNA3.1（ ＋ ） - Bmi-1 was transfected to MDA-MB-468 cell line, and the expression of Bmi-1 mRNA and hTERT mRNA before and after transfection was determined by RT-PCR after 48h. Results： The MCF-7 cell line showed high level of the Bmi-1 mRNA expression, whereas MDA-MB-468 showed modestly increased expression. Bmi-1 gene was cloned from MCF-7 cells, and its eukaryotic expression vector was constructed sucessfully. The espression of Bmi-1 was significantly up-regulated after transfection and its overexpression up-regulated the expression of hTERT mRNA. Conclusion： The expression vector pcDNA3.1（ ＋ ）-Bmi-1 was constructed sucessfully and could be expressed in human mammary carcinoma cell line MDA-MB-468, so as to facilitate the study of the function of Brni-1 in cell lines.

关 键 词：PcDNA3.1(+)-Bmi-1 BMI-1 基因转染 MDA-MB-468 HTERT 

分 类 号：R655.8[医药卫生—外科学]