表达SARS-CoV-2 Delta突变株S蛋白受体结合域和N蛋白的重组腺病毒的构建及鉴定  

Construction and identification of recombinant adenovirus expressing S protein receptor binding domain and N protein of SARS-CoV-2 Delta variant

作　　者：宋泽鑫 周艳[1] 林晓晨 赵永美 施鹏 王学付 张朝武 李鸿钧[1] SONG Zexin;ZHOU Yan;LIN Xiaochen;ZHAO Yongmei;SHI Peng;WANG Xuefu;ZHANG Chaowu;LI Hongjun(Key Laboratory of Vaccine Development for Major Infectious Diseases in Yunnan Province,Institute of Medical Biology,Peking Union Medical College,Chinese Academy of Medical Sciences,Kunming 650118,Yunnan Province,China)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所、云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《中国生物制品学杂志》2023年第9期1025-1031,共7页Chinese Journal of Biologicals

基　　金：云南省应用基础研究计划(202201AT070236);云南省重大科技专项(202202AA100006);中国医学科学院医学与健康科技创新工程重大协同创新项目(2021-I2M-1-043)。

摘　　要：目的构建表达严重急性呼吸综合征冠状病毒2(severe acute respiratory symptom coronavirus 2,SARS-CoV-2)Delta突变株S蛋白受体结合域(receptor binding domain,RBD)和N蛋白的重组腺病毒,并进行鉴定。方法将SARSCoV-2 RBD和N基因片段分别克隆至pcDNA3.0BA载体上,构建重组质粒pcDNA3.0BA-RBD-N,PCR扩增RBD-CMVN片段,连接至穿梭载体pShuttle-CMV上,将穿梭质粒pShuttle-RBD-N与pAdeasy-1进行同源重组,获得重组质粒pAdeasy-1-RBD-N,转染HEK293细胞进行重组腺病毒Ad-RBD-N包装,RT-PCR法检测重组腺病毒中RBD和N基因在HEK293细胞中的转录,Western blot和免疫荧光法检测重组腺病毒中RBD和N蛋白的表达。将Ad-RBD-N通过肌肉注射免疫12只雌性BALB/c小鼠,免疫剂量为5×10^(9)copies/只,免疫后14 d经尾静脉采血,ELISA法检测血清抗体效价。结果重组腺病毒RBD和N基因能在HEK293细胞中正常转录,RBD和N蛋白能在MA104细胞中正常表达。免疫重组腺病毒的小鼠可产生针对RBD和N蛋白的特异性IgG抗体。结论成功构建了表达SARS-CoV-2 Delta突变株S蛋白RBD和N蛋白的重组腺病毒,为Delta突变株疫苗的后续研究奠定了基础。Objective To construct and identify a recombinant adenovirus expressing S protein receptor binding domain(RBD)and N protein of severe acute respiratory symptom coronavirus 2(SARS-CoV-2)Delta variant.MethodsThe RBD and N gene fragments of SARS-CoV-2 were cloned into pcDNA3.0BA vector respectively to construct recombinant plasmid pcDNA3.0BA-RBD-N.The RBD-CMV-N fragment was amplified by PCR and inserted into shuttle vector pShuttle-CMV.The shuttle plasmid pShuttle-RBD-N was then homologously recombined with pAdeasy-1 to obtain recombinant plasmid pAdeasy-1-RBD-N,which was transfected into HEK293 cells for recombinant adenovirus Ad-RBD-N packaging.The transcription of RBD and N genes of recombinant adenovirus in HEK293 cells was detected by RT-PCR,while the expre-ssion of RBD and N proteins by Western blot and immunofluorescence assay.12 female BALB/c mice were immunized with Ad-RBD-N by intramuscular injection at a dose of 5×10^(9)copies per mouse.Blood samples were collected 14 d after immunization,and the serum antibody titers were measured by ELISA.ResultsThe RBD and N genes of recombinant adenovirus were transcribed normally in HEK293 cells,and the RBD and N proteins were expressed normally in MA104 cells.Mice immunized with the recombinant adenovirus produced specific IgG antibodies against RBD and N proteins.ConclusionThe recombinant adenovirus expressing S protein RBD and N protein of SARS-CoV-2 Delta variant was succe-ssfully constructed,which laid a foundation of the follow-up research on Delta variant vaccines.

关 键 词：严重急性呼吸综合征冠状病毒2 S蛋白 受体结合域 N蛋白 腺病毒 

分 类 号：R373.1[医药卫生—病原生物学]