荧光素酶标记的重组人3型腺病毒的构建及分析  

Construction and Analyses of Recombinant Human Adenovirus Type 3 Labeled with Luciferase

作　　者：陈诗颖 周志超[1] 田新贵[1] 周荣[1] CHEN Shiying;ZHOU Zhichao;TIAN Xingui;ZHOU Rong(The First Affiliated Hospital of Guangzhou Medical University,State Key Lab of Respiratory Disease,Guangzhou Medical University,Guangzhou 510182,China)

机构地区：[1]广州医科大学附属第一医院,呼吸疾病国家重点实验室,广州医科大学,广州510182

出　　处：《病毒学报》2020年第4期660-669,共10页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:31570163),题目:人B组腺病毒纤毛蛋白与DSG2受体亲和力的差异及其对病毒致病力的影响研究;国家科技重大专项(项目号:2017ZX10103011003),题目:发热呼吸道感染住院患者病原谱及病原特征研究;广州市科技重大专项(项目号:201803040004),题目:病毒感染重症救治的特异性被动免疫应用研究及腺病毒单克隆抗体药物研发;国家科技重大专项(项目号:2018ZX10102001),题目:临床来源的呼吸道类突发急性传染病病毒分离培养及筛选鉴定;广州市科技计划项目(项目号:201504010032),题目:人腺病毒和流感病毒动物模型的建立及应用研究;广东省自然科学基金项目(项目号:2016A030313572),题目:构建Ad3/TAV fiber knob嵌合型重组体建立人腺病毒重症感染树鼩动物模型。

摘　　要：人3型腺病毒在人DSG2受体转基因鼠体内复制及侵染等生命过程尚不清楚。本研究旨在构建含萤火虫荧光素酶(Firefly luciferase,Fluc)报告基因的复制型重组人3型腺病毒,为可视化重组人3型腺病毒的应用奠定基础。通过PCR扩增萤火虫荧光素酶报告基因,克隆入去掉EGFP基因的人3型腺病毒穿梭质粒pSKA3E3LR(EGFP),经双酶切后与人3型腺病毒骨架质粒pBRAd3-EGFP的PCR扩增片段经重组酶Exnase体外重组的方法,得到中间载体,最后与pBRAd3-EGFP酶切片段连接,得到重组腺病毒质粒pAd3-LUC;转染AD293细胞,将包装成功的重组腺病毒rAd3-LUC纯化并免疫动物,分析重组腺病毒的特性和在小鼠体内的免疫反应。结果显示采用体外重组、酶切连接等方法成功获得到重组人3型腺病毒质粒pAd3-LUC,线性化的pAd3-LUC转染AD293细胞包装拯救得到重组腺病毒rAd3-LUC,观察到细胞病变和检测到荧光素酶的稳定表达,rAd3-LUC免疫小鼠抗血清可以识别并中和重组腺病毒rAd3-LUC(滴度约128~256)。本研究成功构建了E3区缺失并嵌入荧光素酶的复制型重组人3型腺病毒rAd3-LUC,为监控人3型腺病毒在人DSG2受体转基因小鼠体内复制及侵染等生命过程奠定基础。Replication and infection of human adenovirus type 3 in human DSG2 receptor transgenic mice is incompletely understood.We constructed replication-competent recombinant human adenovirus type 3 containing the firefly luciferase reporter gene.The firefly luciferase reporter gene was amplified by polymerase chain reaction(PCR).Human adenovirus type 3 shuttle plasmid pSKA3E3LR(EGFP)with EGFP removed was cloned,after double-enzyme digestion,and recombined with the PCR amplification fragment of human adenovirus type 3 skeleton plasmid pBRAd3-EGFP by Exnase in vitro to obtain a intermediate vector.Finally,it was linked with the pBRAd3-EGFP enzyme-digested fragment to obtain the recombinant adenovirus plasmid pAd3-LUC.The latter was transfected into AD293 cells,and the successfully packaged version was purified and immunized.The characteristics of the recombinant adenovirus and its immune response in mice were analyzed.Results showed that the recombinant human adenovirus type 3 plasmid pAd3-LUC was obtained by in vitro recombination,enzyme digestion and ligation.The linear pAd3-LUC transfected into AD293 cells was packaged to rescue the recombinant virus rAd3-LUC.Cell lesions were observed and stable expression of luciferase was detected.Also,rAd3-LUC mouse antiserum could identify and neutralize the recombinant virus rAd3-LUC(titer of about 128–256).We showed that the replication-competent recombinant human adenovirus type 3 rAd3-LUC absent the E3 region and embedded with luciferase could be constructed.This strategy lays a foundation for monitoring the replication and infection of human adenovirus type 3 in human DSG2 receptor transgenic mice.

关 键 词：人腺病毒(HAdV) 萤火虫荧光素酶(Fluc) 报告基因 构建 免疫反应 

分 类 号：R373.9[医药卫生—病原生物学] Q78[医药卫生—基础医学]