利用Gibson DNA组装技术构建人5型腺病毒感染性克隆

Construction of an Infectious Clone of Human Adenovirus Type 5 Using the Gibson DNA Assembly Method

为了构建人5型腺病毒（HAdV-5）的感染性克隆,我们使用了Gibson DNA组装技术。设计1对引物用于扩增质粒骨架（包含卡那霉素抗性基因和质粒的复制起点,KAN-ORI）,在引物的5’端添加HAdV-5基因组末端序列（约30NT）和Pac I位点,以携带卡那霉素抗性基因的pShuttle-CMV质粒为模板,PCR扩增得到KAN-ORI片段,该片段的两端均含有约30bp的HAdV-5基因组序列。将KAN-ORI片段与利用野毒扩增纯化的HAdV-5基因组混合,进行Gibson DNA组装反应;反应产物直接转化E.coli感受态细胞,涂布含卡那霉素的LB琼脂平板。挑取菌落,提取质粒,命名为pKAd5。限制性酶切鉴定的结果显示,所鉴定的质粒均含有完整的HAdV-5基因组。使用Pac I酶切线性化pKAd5,利用脂质体转染293细胞,转染4d后单层细胞出现病毒噬斑。拯救的病毒能够在HEp-2细胞扩增;电子显微镜观察呈现典型的腺病毒形态;提取病毒DNA,酶切鉴定的结果与预期的HAdV-5基因组相同,证实HAdV-5得到成功拯救。上述研究结果说明使用Gibson DNA组装技术构建HAdV-5感染性克隆简便易行,为其他DNA病毒基因组的克隆提供了新思路。

We attempted to construct an infectious clone of human adenovirus type 5（HAdV-5）using the Gibson DNA assembly method.One pair of primers for amplifying the plasmid backbone was designed and synthesized.Each primer contained three sequence components：a template-specific sequence was added at the 3′end for template priming during polymerase chain reaction（PCR）;an＂overhang＂of 30 nucleotides was added at the 5′end,which was homologous to the terminal sequence of the HAdV-5 genome;and a Pac I restriction site was added between the two components mentioned above.The plasmid backbone（KAN-ORI）was amplified by PCR using aplasmid bearing the kanamycin resistance gene as the template.KAN-ORI contained a kanamycin resistance gene and a plasmid replication origin,which were flanked with30 bp of the HAdV-5 genomic terminal.Gibson DNA assembly was carried out by mixing KAN-ORI and HAdV-5 genomic DNA that was extracted from a purified wild-type HAdV-5 virus.The assembled products were used to transformEscherichia coli-competent cells.Plasmids（pKAd5）were extracted from kanamycin-resistant colonies.Restriction analyses showed that all plasmids contained the complete HAdV-5 genome.PacI-linearized pKAd5 was used to transfect 293 cells,and virus plaques appeared on the cellular monolayer 4 days after transfection.The rescued virus could replicate in HEp-2 cells,and showed typical adenovirus morphology under electron microscopy.The restriction map of viral genomic DNA suggested that the rescued virus was HAdV-5.In conclusion,Gibson DNA assembly facilitated construction of a HAdV-5 infectious clone.Our results suggest the possible application of the Gibson assembly method for cloning viral genomic DNA.