radixin定点突变过表达对hepg2细胞膜转运蛋白bsep的影响 封欣婵 .doc

Radixin定点突变过表达对HepG2细胞膜转运蛋白BSEP的影响 封欣婵，柴 进，程 英，陈文生 （400038 重庆，第三军医大学西南医院全军消化病研究所） [摘要] 目的 构建pcDNA3.1-RDX定点突变真核过表达质粒，研究其在胆汁淤积时对HepG2细胞膜转运蛋白BSEP定位表达的影响。方法 从含有RDX野生型质粒中，利用PCR方法钓取RDX野生型基因片段并以野生型为基础进行定点突变，PCR扩增后转入pcDNA3.1载体，其产物转化DH-5α感受态细胞。对长出的单克隆进行菌落PCR鉴定，再对PCR鉴定阳性的克隆进行测序和比对分析，比对正确即为构建成功的目的质粒。将目的质粒转染HepG2细胞，经G418筛选构建稳转细胞株。提取各株细胞的总蛋白，检测磷酸化RDX是否影响HepG2细胞膜上转运蛋白BSEP的表达。结果 PCR和测序结果均证实pcDNA3.1-RDX WT、pcDNA3.1-RDX T564D、pcDNA3.1-RDX T564ApcDNA3.1-RDX WT、pcDNA3.1-RDX T564D、pcDNA3.1-RDX T564ARDX的磷酸化能增强HepG2细胞膜上BSEP的表达。 [关键词] RDX定点突变；载体构建；pcDNA3.1载体；胆盐输出泵 [中图法分类号] R34；R575.5； Q291 [文献标志码] A The effect of Radixin mutagenesis over-expression on HepG2 cell Feng Xinchan, Chai Jin, Cheng Ying, Chen Wensheng（Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China） [Abstract] Objective To construct and identificate Radixin mutagenesis over-expression vectors-pcDNA3.1-RDX WT, pcDNA3.1-RDX T564D, pcDNA3.1-RDX T564A and transfect the vectors to HepG2 cell. Methods Three different plasmids were cloned to pcDNA3.1 vector and transformed to DH-5α compentent cell. Identify the monoclonal bacteria by PCR method. Wsetern blot to confirm the expression of RDX, p-Thr564-RDX and BSEP. Results Sequences of recombinant plasmid were correct, and the mutants of Radixin were obtained. Western blot demonstrated that compare with RDX wild type and dephosphorylation, the expression of BSEP were increased in HepG2 with RDX phosphorylation(P0.05). Conclusion Successfully constructed Radixin wide-tpye and mutagenesis over-expression vectors and the expression of BSEP were identified, which lay a foundation for a future research. [Key words] Radixin mutagenesis; vector construction; pcDNA3.1; BSEP Supported by the National Science Foundation of China 81170430) and the National Science Foundation of China for the Youth. Corresponding author: Chen Wensheng,E-mail:wenshengchen@ [基金项目] 国家自然科学