间隙连接蛋白connexin43基因在真核细胞中的表达

Expression of gap junction protein connexin43 in eukaryote cells

目的 :构建携带间隙连接蛋白 connexin4 3 c DNA的真核表达载体 ,建立 connexin4 3蛋白高表达细胞株。 方法 :connexin4 3 c DNA在大肠杆菌中扩增后连入真核表达载体 p ED4 ,转染中国仓鼠卵巢 ( CHO)细胞 ,用甲氨蝶呤 ( MTX)筛选得到 connexin4 3基因高表达细胞。结果 :酶切电泳结果显示载体 p ED4 -Cx4 3按设计构建。经 MTX筛选得到不同抗性的 CHO细胞 ,免疫组织化学方法显示有 connexin4 3蛋白的表达 ,但表达量有限。细胞间染料传递实验证实不同 MTX抗性的 CHO细胞间传递小相对分子质量物质的能力有差异。 结论 :利用载体 p ED4可以使 connexin4 3基因在真核细胞内表达。

Objective: : To construct a vector encoding full-length c o nnexin43 cDNA and establish a high connexin-expression cell line. Metho ds: The plasmid encoding full-length connexin43 cDNA was amplified in E. coli RR1. The cDNA was reclaimed from gel and inserted into the eukaryoti c vector pED4. The recombinant transfected Chinese hamster ovary (CHO) cell usua lly expressed low levels of gap junction protein connexin43 and displayed very w eak dye coupling. Stable transfectants were selected by increasing stepwise the concentration of methotrexate (MTX) in culture medium. Results: Electrophoresis results indicated the vector was constructed. Several MTX-resis tance transfected clones were obtained indicating various amounts of connexin43 transcribed from the inserted cDNA. Immunocytochemical analysis revealed an incr ease in the amount of connexin43 immunoreactivity in the transfected cells. The level of dye coupling was also assessed and was found correlated with the amount of connexin43. Conclusion: The connexin43 cDNA inserted into the pE D4 vector can be expressed in the eukaryotic cells.