摘要
目的构建小鼠NeuroD重组腺病毒载体及相应的对照病毒载体，并验证NeuroD基因在小鼠胰岛中的表达活性。方法设计合成一对小鼠NeuroD的eds区的引物序列，以小鼠胰岛cDNA为模板PCR扩增目的片段，凝胶电泳回收DNA片段，经双酶切，将目的片段克隆至AdTrack—CMV穿梭质粒上，并用腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组，挑选阳性克隆小提质粒转染293A细胞．经包装得到pAd—NeuroD和pAd—CMV重组腺病毒，感染小鼠胰岛，Western印迹法检测胰岛内NeuroD蛋白表达水平。结果经验证构建成功带有NeuroD基因的腺病毒载体，能在小鼠胰岛中稳定过量表达。结论成功构建了小鼠胰岛β细胞来源的NeuroD腺病毒载体，为进一步研究NeuroD基因在胰岛β细胞中功能奠定了基础。
Objective To construct an adenovirus vector that over-expresses mouse NeuroD and verify the expression of NeuroD in mouse islets. Methods A pair of specific primers to cds region of mouse NeuroD was designed and the DNA fragment was amplified by PCR, with mouse islet cDNA serving as template. The PCR products were obtained by gel purification. After identified by the ligation reaction, the target fragments were cloned into the AdTrack-CMV shuttle plasmid, which was recombined with back-bone pAdEasy-1 in BJ5183 bacteria. The recombinant pAd-NeuroD adenovirus particles were produced by transfection into QBI-293A cells, and they subsequently infected mouse islets. The NeuroD protein levels were detected by Western blotting. Results The adenoviral vectors were proven successfully constructed with high infection efficiency, and NeuroD was stably over-expressed in mouse islets. Conclusion The recombinant pAd-NeuroD was successfully constructed, which provided a basis for further study of the effect of NeuroD on pancreatic β-cells function.
出处
《医学分子生物学杂志》
CAS
2013年第2期69-72,共4页
Journal of Medical Molecular Biology
