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目的:构建小鼠成红细胞白血病病毒E26癌基因同系物1(V-Ets avian erythroblastosis virus E26 oncogene homolog1,Ets-1)重组腺病毒,并验证其对小鼠原代胰岛及胰岛β细胞系INS-1的感染活性和Ets-1蛋白表达活性。方法:以小鼠胰岛β细胞系MIN6的cDNA为模板,PCR扩增Ets-1基因的编码区序列(coding sequence,CDS)区,纯化后经限制性内切酶切割和连接反应插入到pAdTrack-CMV穿梭质粒上,构建成pAdTrack-CMV-Ets-1质粒。将该质粒与腺病毒骨架质粒pAdEasy-1在大肠杆菌BJ5183中进行同源重组,鉴定出阳性克隆。扩增并抽提阳性克隆质粒,用PacⅠ酶切线性化后转染293A细胞,经包装得到AdV-Ets-1重组腺病毒。相应对照病毒AdV-GFP由相同方法构建。腺病毒经纯化后,感染小鼠原代胰岛及INS-1细胞,并用Western blot检测Ets-1蛋白表达水平。结果:腺病毒构建成功,并具有高感染率及高蛋白表达能力。结论:成功构建了小鼠Ets-1腺病毒,为进一步研究Ets-1基因在原代胰岛中的功能提供基础。
OBJECTIVE: To construct the recombinant adenovirus of mouse E-leukemia virus E26 oncogene homolog 1 (Ets-1) and to investigate its effect on the expression of mouse primary islets and pancreatic islet β cell line INS- 1 infection activity and Ets-1 protein expression activity. Methods: The CDS region of Ets-1 gene was amplified by PCR using the cDNA of mouse pancreatic β-cell line MIN6 as a template. After purification, the coding region sequence of Ets-1 gene was amplified by restriction endonuclease digestion and inserted into pAdTrack- CMV shuttle plasmid to construct pAdTrack-CMV-Ets-1 plasmid. The plasmid and adenoviral backbone plasmid pAdEasy-1 in E. coli BJ5183 homologous recombination identified positive clones. The positive clone plasmids were amplified and extracted. The recombinant plasmids were linearized with Pac I and transfected into 293A cells to obtain AdV-Ets-1 recombinant adenovirus. The corresponding control virus AdV-GFP was constructed by the same method. After being purified, the adenovirus was infected into primary islets of mice and INS-1 cells, and the expression of Ets-1 protein was detected by Western blot. Results: The adenovirus was constructed successfully with high infection rate and high protein expression. CONCLUSION: The mouse Ets-1 adenovirus was successfully constructed and provided the basis for further study on the function of Ets-1 gene in primary islets.