pEGFPBMI1真核表达载体构建、鉴定及其表达.docVIP

pEGFPBMI1真核表达载体构建、鉴定及其表达 　论文联盟编辑。 作者：陈凤花，胡丽华，王琳，李一荣 论文代写 【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％ (Plt;0.01)。结论： 成功构建了真核表达质粒pEGFPBMI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。 【关键词】 基因转染 Construction, Identification and Expression of Recombinant Eukaryotic Vector pEGFPBMI1 Abstract 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 GreenⅠrealtime RTPCR was used to quantitate P16INK4a 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 P16INK4a mRNA expression was reduced to 9.2%. It is concluded 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. 毕业论文 Key words pEGFPBMI1; BMI1; gene transfection; HeLa cell; P16INK4a 毕业论文 J Exp Hematol 2007; 15(5):1056-1060 PcG (polycomb group， PcG)基因家族以多蛋白复合体的形式调控靶基因的表达［1］，无论在果蝇还是哺乳动物的定向发育以及细胞的增