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cds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 re -gion 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 o-ver-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.
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