海人藻酸受体GluK2亚基复制缺陷型腺病毒的构建  

Construction of Replication- deficient Adenovirus Recombinants for Kainate Receptor Subunit GluK2

作　　者：杜彩萍[1] 徐镇[1] 胡书群[1] 李婷[1] 侯筱宇[1] 

机构地区：[1]徐州医学院,江苏省脑病生物信息重点实验室,生物化学与分子生物学研究中心,江苏徐州221004

出　　处：《生物技术》2014年第1期12-16,共5页Biotechnology

基　　金：国家自然科学基金项目("脑缺血PSD-95酪氨酸磷酸化对突触后Src信号网络的调控";编号:81173030;"MLK3的SUMO化修饰在缺血性脑损伤中的作用";编号:81100852);江苏省高校自然科学研究重大项目("脑缺血蛋白质类泛素SUMO化修饰的研究";编号:11KJA310005);江苏省高校优势学科建设工程资助项目(PAPD);江苏省青蓝工程;"徐州医学院振兴计划"资助~~

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。Objective： To construct replication - deficient adenovirus recombinants for kainate receptor subunits GluK2. Method： GluK2 cDNAs were obtained by PCR from GluK2 -peDNA3.1 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 electropora- tion 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 purifi- cation followed by titer determination. Result：GluK2 adenovirus present strong infection ability （6. 75 × 10^9 ifu/ml） and can express the target protein. Conelusion：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.

关 键 词：GluK2 海人藻酸受体 腺病毒 复制缺陷型 感染能力 

分 类 号：Q784[生物学—分子生物学]