【摘要】 目的:构建海人藻酸(kainate,KA)受体GluK2亚基复制缺陷型腺病毒重组体。方法:以GluK2-pcDNA3.1真核表达载体为模板,经PCR扩增GluK2目的基因,然后将其亚克隆入腺病毒穿梭载体pAdTrack-CMV,后将重组的穿梭质粒与腺病毒骨架载体pAdEasy-1共同电转入BJ5183电穿孔感受态细胞筛选阳性同源重组子。阳性重组体经限制性内切酶PacⅠ消化,经脂质体转染入HEK293细胞进行包装与扩增。接着腺病毒经大量提取纯化后进行滴度测定。结果:GluK2腺病毒具有较强的感染能力(6.75×109ifu/ml)并可表达目的蛋白。结论:GluK2复制缺陷型腺病毒成功构建。GluK2腺病毒可高效感染原代神经元及神经细胞系,为进一步研究其对神经元的调控作用奠定基础。更多还原

【Abstract】 Objective: To construct replication- deficient adenovirus recombinants for kainate receptor subunits GluK2. Method: GluK2 cDNAs were obtained by PCR from GluK2- pcDNA3. 1 eukaryotic expression vector,and then cloned into the adenovirus shuttle vector pAdTrack- CMV. Then the recombinant shuttle plasmid and backbone vector pAdEasy- 1 were co- transformed into BJ5183 electroporation component cells to screen positive homologous recombinant. The positive recombinant was digested by restriction endonuclease Pac Ⅰ, and then transfected into HEK293 cells for packaging and amplification. The adenovirus was then sent to large quality extraction and purification followed by titer determination. Result: GluK2 adenovirus present strong infection ability( 6. 75 × 109ifu / ml) and can express the target protein. Conclusion: GluK2 replication- deficient adenovirus recombinants were constructed successfully. GluK2 adenovirus could infect primary neurons and neuronal cell lines efficiently,which provide significance for studying the role of GluK2 in neuronal regulation.更多还原