文献类型：期刊文献

中文题名：Ⅲa基因缺失的人7型腺病毒载体系统的建立

英文题名：Establishment of A Ⅲa-Deleted Human Type-7 Adenoviral Vector System

作者：王洋[1,2];王永进[2];侯文哲[2];郭小娟[2];郑贵森[1];邹小辉[2]

第一作者：王洋

机构：[1]甘肃中医药大学公共卫生学院,兰州730000;[2]中国疾病预防控制中心病毒病预防控制所,国家卫生健康委员会医学病毒和病毒病重点实验室,北京100052

第一机构：甘肃中医药大学公共卫生学院

年份：2024

卷号：40

期号：2

起止页码：365

中文期刊名：病毒学报

外文期刊名：Chinese Journal of Virology

收录：CSTPCD;;北大核心:【北大核心2023】；CSCD:【CSCD2023_2024】；PubMed;

基金：国家重点研发计划(项目号:2022YFC2602202),题目:国家生物安全实物核心资源库支撑关键技术研究。

语种：中文

中文关键词：Ⅲa基因;人7型腺病毒载体;单周期载体;DNA组装;病毒拯救

外文关键词：Ⅲa gene;Human adenovirus type-7 vector;Single-cycle vector;DNA assembly;Virus rescue

摘要：为了获得既能高效扩增外源基因,又不产生复制型子代病毒的腺病毒载体,我们构建了一个Ⅲa基因缺失的单周期腺病毒载体。本研究在前期已构建的E3区携带绿色荧光蛋白GFP的人7型腺病毒载体pK-Ad7E3GFP基础上进行。首先设计引物扩增包含氨苄青霉素抗性基因和质粒复制起点的AMP-ORI片段,利用限制性内切酶Asc I将pK-Ad7E3GFP切割为29.1kb与7.7kb的大小片段,其中,7.7kb小片段与AMP-ORI片段组装,获得第一个中间质粒pA-Ad7E3GAscI;经重叠延伸PCR、酶切、DNA组装等方法,将pA-Ad7E3GAscI上的Ⅲa基因进行删除,获得第二个中间质粒pA-AscDⅢa;经Pme I酶切的pA-AscDⅢa与上述29.1kb大片段经DNA组装后,得到重组腺病毒质粒pK-Ad7DⅢaE3GFP。同时,构建携带Ⅲa基因的真核表达载体pTPL-Ⅲa,转染293细胞,经G418筛选构建细胞系293-Ⅲa。结果发现,经Pme I线性化的pK-Ad7DⅢaE3GFP质粒转染293-Ⅲa细胞,7d后可见荧光聚集,且逐渐增大,表明重组腺病毒Ad7DⅢaE3GFP拯救成功;提取病毒基因组进行PCR鉴定,结果与预期相符;细胞培养上清经负染后,在电镜下可见腺病毒典型的正二十面体形态,培养细胞中也可见病毒呈晶格排列;以上结果均表明Ad7DⅢaE3GFP拯救成功。本研究成功建立了Ⅲa基因缺失的单周期人7型腺病毒载体系统,为该载体的研究与应用奠定基础。
We wished to obtain an adenoviral vector that can efficiently amplify exogenous genes without producing replicative progeny viruses.We developed a"single cycle"adenoviral vector with one deletion of the Ⅲa gene.This study was conducted on the basis of pK-Ad7E3GFP,a previously constructed human adenovirus type-7 vector carrying green fluorescent protein(GFP)in the E3 region.First,primers were designed to amplify the AMP-ORI fragment containing the ampicillin-resistance gene and the plasmid origin of replication.pK-Ad7E3GFP was digested with restriction enzyme Asc I to generate two fragments of 29.1 kb and 7.7 kb in size.Then,the 7.7-kb fragment was assembled with the AMP-ORI fragment to produce the first intermediate plasmid pA-Ad7E3GAscI.Using overlap-extension polymerase chain reaction(PCR),enzyme digestion,and DNA assembly,the Ⅲa gene on pA-Ad7E3GAscI was removed,and the second intermediate plasmid pAAscD Ⅲa was produced.The plasmid pA-AscD Ⅲa was digested with Pme I,which was mixed with the 29.1-kb fragment by Gibson DNA assembly,to construct the recombinant adenoviral plasmid pK-Ad7D ⅢaE3GFP.Meanwhile,the eukaryotic expression vector pTPL-II a carrying the Ⅲa gene was constructed and transfected into 293 cells to obtain stably transfected 293-Ⅲa cells by G418 screening.Pme I-linearized pK-Ad7D ⅢaE3GFP was used to transfect 293-Ⅲa cells.GFP foci were observed on monolayer cells 7 days after transfection,suggesting rescue of the Ad7DIl aE3GFP recombinant virus.The viral genome was extracted for identification by PCR,and the results were as expected.The typical icosahedral morphology of adenovirus was seen under an electron microscope after negative staining of cell-culture supernatants.The virus was also seen in a lattice arrangement in cultured cells.All of these observations suggest rescue of Ad7D Ⅲ AE3GFP.We established a single-cycle human type-7 adenoviral vector with deletion of the Ⅲa gene.Our data could lay the foundation for the research and application of this vector.