MIIP真核表达载体的构建及其稳定过表达MDA-MB-231细胞系的建立

Construction of Eukaryotic Expression Vector of MIIP and Establishment of Its Stably Transfected MDA-MB-231 Cell Line

目的构建pCDNA3.0-MIIP真核表达载体并建立其稳定转染的MDA-MB-231细胞系。方法pGEX-4T-1-MIIP重组质粒及pCDNA3.0真核表达载体分别经Xho I和EcoR I双酶切后,回收目的基因,T4 DNA连接酶连接后,转化得到pCDNA3.0-MIIP重组真核表达载体,对其进行双酶切和测序鉴定。脂质体法将鉴定后的pCDNA3.0-MIIP质粒转染至人乳腺癌MDA-MB-231细胞中,分别于转染后48和72h提取细胞总RNA,QRT-PCR法确定其转染。G418筛选转染细胞,Western blot检测目的蛋白表达。结果酶切及测序结果证实pc DNA3.0-MIIP真核表达载体构建成功。pcDNA3.0-MIIP转染至MDA-MB-231细胞后,QRT-PCR检测证实MIIP表达显著增强。G418筛选后得到单克隆细胞群,Western blot检测证实MIIP表达显著增强。结论成功构建出了可在真核细胞中高效表达MIIP基因的pc DNA3.0-MIIP重组质粒;建立了稳定过表达MIIP的MDA-MB-231细胞系。

Objective To construct the pcDNA3. 0-MIIP eukaryotic expression vector and to establish its stably transfected MDA-MB-231 cell line. Methods The p GEX-4T-1-MIIP recombinant plasmid and pcDNA3. 0 eukaryotic expression vector were digested by Xho I and EcoR I and then the fragments of MIIP gene and pcDNA3. 0 vector were then joined together by T4 DNA ligase,and transformed to host cells to obtain the recombinant eukaryotic expression vector pcDNA3. 0-MIIP. p CDNA3. 0-MIIP was transfected into human breast cancer cell line MDA-MB-231 by lipofectanmine2000,after 48 h and 72 h,total RNA of the cells was extracted and analyzed by QRT-PCR to determine the expression level of MIIP and evaluate the efficiency of transfection. Transfected MDA-MB-231 cell line was selected by G418,and western blot was used to confirm the high expression of MIIP in these cells. Results The eukaryotic expression vector of pcDNA3. 0-MIIP was constructed successfully,which was identified by restriction enzyme digestion and nucleotide sequencing. MIIP was efficiently expressed in pcDNA3. 0-MIIP transfected MDA-MB-231 cells,tested by QRT-PCR. After G418 selection,the obtained monoclonal cell population from pcDNA3. 0-MIIP transfected MDA-MB-231 cells showed a noticeably increased expression of MIIP compared to that from the empty vector transfected cells. Conclusion The eukaryotic expression vector of MIIP gene was constructed successfully,and its stably transfected MDA-MB-231 cell line was established.