目的 构建携带Ubc9基因的重组腺病毒质粒pAdEasy-1/Ubc9,制备含13bc9基因的重组腺病毒Ad-Ubc9,并使Ubc9在HeLa细胞中高效表达.方法 PCR法扩增目的 Ubc9基因:用pemⅠ酶切将穿梭质粒pAdTrack-CMV-Ubc9线性化;将线性化的穿梭质粒pAdTrack-CMV-Ubc9与腺病毒骨架质粒pAdeasy-1在感受态BJ5183菌内进行同源重组,筛选阳性重组子pAdEasy-1/Ubc9;再用PacⅠ酶切pAdEasy-1/Ubc9使之线性化,线性化的重组腺病毒质粒经脂质体转染HEK293细胞,进行重组腺病毒的包装和扩增.收集重组腺病毒,感染HeLa细胞,用Western blot方法检测Ubc9在HeLa细胞中的表达.结果 通过Pac Ⅰ酶切证实携带Uboc9基因的腺病毒载体构建成功,包装出携带Uboc9基因的腺病毒能有效感染HeLa细胞.结论 利用细菌内同源重组方法成功地构建了携带Ubc9的重组腺病毒载体,并能在HeLa细胞中高效表达.%Objective To construct the recombinant adenovirus plasmid containing Ubc9 gene pAdEasy-1/ Ubc9, prepare recombinant adenovirus Ad-Ubc9 and efficiently express the Ubc9 gene in HeLa cells.Methods pAdTrack-CMV-Ubc9 was linearized with PmeI and transformed into ultracompletent BJ5183 containing pAdeasy-1 The recombinant adenovirous plasmid pAdEasy-1/Ubc9 was then constructed by homologous recombination in bacteria BJ5183, and was identified by PacI digestion.Linearized pAdEasy-1/Ubc9 was then transfected into HEK 293 cells with liposome to generate recombinant adenovirus particles used to infect HeLa cells.Results After HEK293 cells were transfected with recombinant adenovirus the RFP fluorescence could be observed.The expression of Ubc9 protein was high in HeLa cells detected by western blotting.Conclusion The recombinant adenovirus vector pAdEasy-1/Ubc9 was constructed successfully and the Ubc9 gene could be effectively expressed in HeLa cells.
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