重组腺病毒Ad-NLS-RARα的构建及其在K562细胞中的表达

Construction of recombinant adenovirus Ad-NLS-RARα and its expression in K562 cells

目的构建含NLS-RARα基因的重组腺病毒Ad-NLS-RARα,并检测其在K562细胞中的表达。方法以质粒pGBKT7-PML-RARα为模板,PCR扩增NLS-RARα基因,与穿梭质粒dTrace-TO4连接,构建重组穿梭质粒pAdTrack-TO4-NLS-RARα,经PmeⅠ酶切线性化后,转化含腺病毒骨架质粒pAdEasy-1的感受态大肠埃希菌BJ5183,获得重组腺病毒质粒pAd-NLS-RARα,经PacⅠ酶切线性化后,转染AD293细胞,获得重组腺病毒Ad-NLS-RARα,经4轮扩增后,测定重组腺病毒滴度,并进行PCR及测序鉴定;将重组腺病毒感染K562细胞,采用流式细胞术测定感染效率,RT-PCR法和Western blot法检测NLS-RARα在K562细胞中的转录及表达水平。结果重组腺病毒Ad-NLS-RARα经4轮扩增后,滴度可达6.9×108pfu/ml,PCR及测序鉴定证明构建正确,对K562细胞的感染效率可达70%左右;重组腺病毒Ad-NLS-RARα携带的NLS-RARα基因可在K562细胞中高效表达。结论已成功构建了重组腺病毒Ad-NLS-RARα,并在K562细胞中高效表达了NLS-RARα基因。

Objective To construct recombinant adenovirus Ad-NLS-RARα and determine its expression in K562 cells.Methods NLS-RARα gene was amplified by PCR using plasmid pGBKT7-PML-RARα as a template and inserted into shuttle plasmid dTrace-TO4.The constructed recombinant shuttle plasmid pAdTrack-TO4-NLS-RARα was digested with PmeⅠ,and transformed to competent E.coli BJ5183 containing adenovirus backbone plasmid pAdEasy-1.The obtained recombinant adenovirus plasmid pAd-NLS-RARα was digested with PacⅠ and transfected to AD293 cells for packaging.The obtained recombinant adenovirus Ad-NLS-RARα was subjected to four cycles of amplification,determined for titer and identified by PCR and sequencing.K562 cells were infected by the recombinant adenovirus,then determined for infection efficacy by flow cytometry,and for mRNA transcription and protein expression of NLS-RARα by RT-PCR and Western blot respectively.Results Recombinant adenovirus Ad-NLS-RARα reached a titer of 6.9 × 108 pfu / ml after four cycles of amplification and was constructed correctly as proved by PCR and sequencing,with which the infection efficacy of K562 cells was about 70%.The NLS-RARα gene in Ad-NLS-RARα was highly expressed in K562 cells.Conclusion Recombinant adenovirus Ad-NLS-RARα was constructed successfully,and NLS-RARα gene was highly expressed in K562 cells.