重组质粒pEGFP-mDlx5的构建及其在SVZa神经干细胞中的表达研究  

Construction and expression of recombinant plasmid pEGFP-mDlx5 in neural stem cells derived from anterior subventricular zone of neonatal rats

作　　者：高方友[1] 杨辉[1] 张治元[1] 何家全[1] 刘仕勇[1] 邱克军[1] 

机构地区：[1]中国人民解放军第三军医大学新桥医院神经外科,重庆400037

出　　处：《中华神经医学杂志》2006年第5期450-453,共4页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金重点项目(30130110)

摘　　要：目的构建真核表达重组质粒pEGFP-mDlx5,并了解它在室管膜前下区(SVZa)神经干细胞(NSCs)中的mRNA及融合蛋白表达情况。方法运用DNA重组技术自原核表达载体上将小鼠来源的mDlx5基因克隆到增强型绿色荧光蛋白(EGFP)载体上,并用双酶切、测序进行鉴定;将重组质粒用电穿孔的方法转染SVZa NSCs,荧光显微镜下动态观察荧光变化情况;培养24h后提取转染细胞的总RNA及总蛋白,通过逆转录-多聚酶链反应(RT-PCR)了解其mRNA表达情况,用Western blot 检测其蛋白表达情况。结果重组质粒通过双酶切产生了0．78 kb目的插入片段及4．68 kb载体片段;测序后证实0．78 kb片段碱基序列与mDlx5基因完全同源;重组质粒转染SVZa NSCs 8 h后在荧光显微镜下观察到绿色荧光,12 h后逐渐增多,24-48 h达高峰,且稳定表达较长时间;24 h后通过 RT-PCR检测到mRNA表达,Western blot检测到58 kD目的蛋白表达。结论新构建的真核表达重组质粒pEGFP-mDlx5通过鉴定,结构正确:转染到SVZa NSCs后能在其中表达、发挥功能,为后续研究奠定了基础。Objective To construct an eukaryotic expression recombinant plasmid named pEGFP-mDlx5 and get the message of its mRNA transcription and protein translation in in vitro neural stem cells（NSCs） derived from the anterior subventricular zone （SVZa） of the neonatal rats. Methods Mouse Dlx5 gene base sequence derived from prokaryotic expression vector BluescriptBSK-mDlx5 was inserted to multiple clone sites of EGFP-C vector by DNA recombinant technique. Then a new eukaryotic expression recombinant plasmid named pEGFP-mDlx5 was generated and identified by incision enzyme EcoR I and Kpn I and DNA sequencing. PEGFP-mDlx5 was transfected into SVZa NSCs by electroporation, from which total RNA and protein were extracted after 24 h for detecting their expressions by reverse transcriptase-polymerase chain faction （RT-PCR） and Western blot analysis. The treated cells were continuously traced by fluorescence microscope. Results The recombinant plasmid cut by incision enzyme EcoR I and Kpn I overnight generated a 0.78 kb fragment and a 4.68 kb fragment in 1% agarose gel electrophoretogram. And DNA sequence of the 0.78 kb fragment was identical with mouse Dlx5 mRNA in GenBank. In the cells 24 h after transfected with recombinant plasmid, RT-PCR revealed that mDlx5 mRNA was expressed, and Western blot analysis showed a 58 kD fusion protein. Green fluorescence could be seen by fluorescence microscope after 8 h and a peak emerged within 24 to 28 h. Conclusion The recombinant plasmid was successfully constructed and is a useful tool to research the migration and differentiation of SVZa NSCs.

关 键 词：重组质粒 绿色荧光蛋白 D1x5基因 室管膜前下区 神经干细胞 

分 类 号：Q78[生物学—分子生物学]