Neurexin 1β及其剪切变异体真核表达载体构建  

Construction of Neurexin 1β and its splice variant eukaryotic expression plasmid vector

作　　者：徐岩[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》2016年第1期1-5,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”,No.81173030);江苏省高校优势学科建设工程资助项目(PAPD)资助

摘　　要：[目的]构建大鼠Neurexin 1β(Nrx 1β)及其剪切变异体Nrx 1β(ΔS4)真核表达载体,并鉴定其在真核细胞中的表达能力。[方法]以提取的大鼠海马总RNA为模板,采用RT-PCR技术获得Nrx 1β及Nrx 1β(ΔS4)目的基因,通过分子生物学方法将目的基因亚克隆至p DsRed2-C1载体。重组体转染HEK293T细胞,免疫印迹检测Nrx 1β及Nrx1β(ΔS4)的蛋白表达。[结果]Nrx 1β及Nrx 1β(ΔS4)目的基因扩增成功,重组体酶切鉴定能观察到目的基因片段(约1500 bp处),Nrx 1β及Nrx 1β(ΔS4)测序结果与Gen Bank中的序列一致。免疫印迹显示,p DsRed2-C1-Myc-Nrx 1β或p DsRed2-C1-Myc-Nrx 1β(ΔS4)在HEK293T细胞中能表达Nrx 1β或Nrx 1β(ΔS4)。[结论]成功构建大鼠Nrx1β及其剪切变异体Nrx 1β(ΔS4)的真核表达载体,重组体在HEK293T细胞中高效表达。[ Objective ] To construct the recombinant of rat Neurexin 1β （Nrx 1β） and its splice variant eukaryotic expression plasmid Nrx 1β （ΔS4）, and to identify their expression ability in eukaryotic cells. [ Methods] Target genes of Nrx 1β and Nrx 1β （ΔS4） were expanded by RT -PCR using rat hippocampal total RNA as template. Then the target genes were sub -cloned into the pDsRed2 - C1 vector by molecular biology method. The recombinants were transfected into HEK293T cells, and protein expression was detected by Western Blot. [ Results ] Target genes of rat Nrx 1β and Nrx 1 ~ （ΔS4） were successfully amplified. The recombinants were digested with restriction enzyme and proved to have the target genes （ about 1 500 bp）. The results of Nrx ! 13 and Nrx 1β （ΔS4） sequencing were consistent with the gene sequence in GeneBank. Western Blot analysis showed that pDsRed2 - CI - Myc - Nrx 1β or pDsRed2 - CI - Mye - Nrx 1β （ΔS4） expressed Nrx 1β and Nrx 1β （ΔS4） in HEK293T cells. [ Conclusion] The eukaryotic expression plasmids of rat Nrx 1β and its splice variant Nrx 1β （ΔS4） were successfully constructed, and were highly expressed in HEK293T cells.

关 键 词：NEUREXIN 1β 剪切变异体 真核表达载体 蛋白表达 

分 类 号：Q784[生物学—分子生物学]