利用限制性酶切-组装方法构建携带双报告基因的猴1型腺病毒载体  

Construction of A Simian Adenovirus 1 Vector Carrying Dual-Reporter Genes by Restriction-Assembly

作　　者：陈娟 邹小辉[2] 郭小娟[2] 王晓晖 鲁茁壮[2] CHEN Juan;ZOU Xiaohui;GUO Xiaojuan;WANG Xiaohui;LU Zhuozhuang(School of Public Health,Baotou Medical College,Inner Mongolia University of Science and Technology,Baotou O14040,China;NHK Key Laboratory of Medical Virology and Viral Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease ControlandPrevention,Beijing100052,China)

机构地区：[1]内蒙古科技大学包头医学院公共卫生学院,包头014040 [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

出　　处：《病毒学报》2023年第3期791-799,共9页Chinese Journal of Virology

基　　金：国家自然科学基金(国际合作与交流项目)(项目号:82161138001),题目:基于肠道腺病毒载体平台COVID-19黏膜疫苗的设计与筛选。

摘　　要：限制性酶切-组装是构建和改造腺病毒载体的一种简便方法,本研究用该方法构建同时携带GFP和萤火虫荧光素酶(Firefly Luciferase, Fluc)报告基因的猴1型腺病毒(Simian Adenovirus 1, SAdV-1)载体。PCR分别扩增GFP和Fluc基因,重叠延伸PCR将两个目的基因通过T2A短肽编码序列串联得到融合基因GFluc。限制性内切酶Spe I酶切腺病毒质粒pKSAV1-EG,去除原有的外源基因,将载体片段与GFluc片段进行限制性酶切-组装,产物转化大肠杆菌感受态细胞,获得腺病毒质粒pKSAV1-GFluc。通过限制性内切酶酶切分析、PCR产物测序证实质粒构建正确。pKSAV1-GFluc质粒经Swa I酶切线性化后转染SAdV-1包装细胞系293SE13,4 d后GFP+细胞形成荧光灶,并持续增大,表明重组病毒拯救成功;对重组病毒基因组进行了限制性酶切鉴定。扩增病毒并进行纯化,在细胞水平和动物模型中检测到目的蛋白GFP和Fluc的表达。本研究成功构建同时携带GFP和Fluc报告基因的SAdV-1载体,显示了限制性酶切-组装方法的易用性和可靠性,并为研究SAdV-1载体在小鼠模型的组织分布奠定基础。Restriction-Assembly is a simplified procedure for construction or modification of adenoviral vectors.In this study,a simian adenovirus 1(SAdV-1)vector carrying GFP and firefly luciferase(Fluc)reporter genes was constructed by using restriction-assembly.GFP and Fluc fragments were amplified by PCR,and overlap extension PCR was performed to link both through T2A short peptide coding sequence to form a fusion gene GFluc.SAdV-1 adenoviral plasmid pKSAV1-EG was digested with restriction enzyme Spel to remove the original exogenous gene.The linearized pKSAV1-EG was combined with GFluc and subjected to Gibson assembly reaction.The assembly product was transformed into Escherichia coli TOP 10 chemically competent cells to generate pKSAV1-GFluc plasmid,which was identified by restriction analysis.Swal-linearized pKSAV1-GFluc was used to transfect 293SE13 packaging cells.GFP foci were seen on the monolayer cells 4 days post transfection,suggesting a successful rescue of SAdV1-GFluc recombinant virus.SAdV1-GFluc was amplified in 293SE13 cells and purified by density gradient ultracentrifugation.HEp-2 cells were infected with SAdV1-GFluc at MOIs of 100,500 or 2500 vp/cell,and GFP+cells were observed under fluorescence microscope and assayed by flow cytometry 2 days post infection.Luciferase activity was also detected in virus-infected HEp-2 cells and mouse.These data indicated that restriction-assembly was an easy-to-use and effective method for adenoviral vector construction.SAdV1-GFluc could be used to study the distribution of transgene in SAdV-1 vector-infected mice.

关 键 词：报告基因 限制性酶切-组装 猴1型腺病毒 载体 

分 类 号：R373.9[医药卫生—病原生物学]