pEGFP-BMI-1真核表达载体的构建、鉴定及其表达  

Construction,Identification and Expression of Recombinant Eukaryotic Vector pEGFP-BMI-1

作　　者：陈凤花[1] 胡丽华[1] 王琳[1] 李一荣[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院检验科,武汉430022

出　　处：《中国实验血液学杂志》2007年第5期1056-1060,共5页Journal of Experimental Hematology

基　　金：湖北省科技攻关项目;编号2005AA304B08

摘　　要：本研究构建真核表达载体pEGFP-BMI-1并观察其在HeLa细胞中的表达。将BMI-1逆转录聚合酶链反应(RT-PCR)产物克隆入pEGFP-N1,经酶切、PCR鉴定及测序分析,构建pEGFP-BMI-1真核表达载体;采用脂质体转染法,将融合基因pEGFP-BMI-1转入HeLa细胞,用荧光显微镜和Western blot检测其表达,SYBR Green I实时定量RT-PCR方法检测转染前后HeLa细胞中P16INK4a mRNA的表达变化。结果表明:重组后的pEGFP-N1质粒已成功载入BMI-1的全长编码基因,序列测定的结果与预期设计完全一致。荧光显微镜下可见在转染pEGFP-BMI-1的HeLa细胞中存在荧光分布;Western blot检测发现存在外源性融合蛋白BMI-1-EGFP的表达。在HeLa细胞中过表达BMI-1显著下调P16INK4a mRNA的表达为对照组的9.2%(P<0.01)。结论:成功构建了真核表达质粒pEGFPBMI-1,转染HeLa细胞后可表达融合蛋白BMI-1-EGFP。这为今后研究BMI-1在肿瘤发生发展中的机制奠定了基础。This study was purposed to construct and identify the mammalian expression vector of pEGFP-BMI-1 and to detect whether it could express in human cervix cancer cell line HeLa. The cDNA fragment of BMI-1 obtained by RT- PCR was inserted into pEGFP-N1. The recombinant plasmid was confirmed by restriction enzyme digestion, PCR and DNA sequencing, pEGFP-BMI-1 was transfected into HeLa cells with lipofectamine 2000. The expression of pEGFP- BMI-1 was determined by EGFP fluorescence and Western blot analysis. SYBR Green I real-time RT-PCR was used to quantitate P16^INK4a mRNA. The results showed that the correct construction of the recombinant plasmid pEGFP-BMI-1 has been shown by restriction enzyme digestion, PCR and DNA sequencing, pEGFP-BMI-1 could express BMI-1-EGFP fusion protein in HeLa cells. Real-time RT-PCR showed that P16^INK4a mRNA expression was reduced to 9.2%. It is con- cluded that the vector of pEGFP-BMI-1 has been successfully constructed and it can be expressed in HeLa cells. This work has laid foundations for further study on biological functions and potential application of BMI-1.

关 键 词：pEGFP—BMI-1 BMI-1 基因转染 HELA细胞 P16^INK4A 

分 类 号：Q782[生物学—分子生物学] R730.231[医药卫生—肿瘤]