重组Mash1慢病毒表达载体的构建

Construction of the Lentiviral Expression Vector Carrying Mash1 Gene

目的:克隆小鼠Mash1基因的全长cDNA,构建其慢病毒表达载体Lentivirus-Mash1,为进一步研究Mash1在神经发育及干细胞神经分化中的作用奠定基础。方法:应用RT-PCR从小鼠13d胚胎中扩增出Mash1cDNA片段,经回收纯化与pGEM-T载体连接并转化感受态细菌J M109,通过蓝白筛选、PCR扩增和双酶切鉴定出阳性菌落,并对其进行基因测序。BamHⅠ和XholⅠ双酶切pGEM-T-Mash1和Lentivirus获得的目的基因双粘片段与Lentivirus双粘线性质粒相连接,转化J M109,酶切方法鉴定重组阳性菌落。结果:PCR扩增、酶切分析及序列测定证实成功获取Mash1cDNA克隆;酶切鉴定证实Lentivirus-Mash1含有大小正确的正向Mash1cDNA片段。结论:成功建立了小鼠Mash1cDNA克隆并构建了慢病毒表达载体Lentivirus-Mash1。

Objective：To construct the lentiviral expression vector of murine Mashl for further study on neural development and neural differentiation. Methods：The entire Mashl cDNA was amplified by RT-PCR from a mouse embryo and then was ligated with pGEM-T vector. The ligation product was transformed into the JM109 competent cells. The positive recombinant clones were selected and identified by α-complementation, PCR, restriction endonuclease digestion and DNA sequencing. The cloning vector and the Lentivirus were cut by BamH Ⅰ and Xhol Ⅰ . Then they were ligated and transformed. The enzyme analysis was used to confirm the recombinant vector. Results;The results of PCR, enzyme analysis and DNA sequencing analysis have confirmed that the right Mashl gene was cloned. Conclusion： The cDNA clone of murine Mashl and its lentiviral expression vector Lentivirus- Mashl are successfully constructed.