表达呼吸道合胞病毒融合前F蛋白的重组人5型腺病毒的构建  

Construction of recombinant human adenovirus type 5 expressing prefusion F protein of respiratory syncytial virus

作　　者：马亚林 柴鹏弟 王金冬[3] 毛彤瑶 张鹏 李慧莹[2] 段招军[2] MA Ya-lin;CHAI Peng-di;WANG Jin-dong;MAO Tong-yao;ZHANG Peng;LI Hui-ying;DUAN Zhao-jun(School of Public Health,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;NHC Key Laboratory of Medical Virology and Viral Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Weifang Medical University,Weifang 261000,China)

机构地区：[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,北京102206 [3]潍坊医学院,潍坊261000

出　　处：《中国人兽共患病学报》2023年第6期515-522,共8页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.21934005)。

摘　　要：目的筛选并构建成功表达A亚型人呼吸道合胞病毒(Human Respiratory Syncytial Virus,HRSV)融合前构象F蛋白的重组人5型腺病毒(Recombinant Human type5 adenovirus,rHADV-5),为制备和研发HRSV重组腺病毒载体疫苗奠定基础。方法在真核表达载体pcDNA3.1(+)上插入具有不同突变位点的HRSV F蛋白编码序列,利用融合前F蛋白单克隆抗体AM14和D25筛选HRSV F蛋白突变体;通过同源重组将筛选出的前构象F蛋白突变体编码序列插入非复制型人5型腺病毒载体,在HEK293细胞中进行重组病毒的包装和扩增,经氯化铯密度梯度法获得纯化的重组病毒;应用夹心ELISA法和Western blot鉴定F蛋白的表达及构象。结果通过AM14和D25单克隆抗体筛选出了2个稳定维持在融和前构象的HRSV F蛋白序列(SC-3M和SC-5M)。将SC-3M和SC-5M编码序列插入非复制型人5型腺病毒载体,对两种重组人5型腺病毒(pAd5-SC-3M和pAd5-SC-5M)和空载体(pAd5-vector)纯化,病毒滴度分别达到2.016×10^(10),2.52×10^(10)和2.948×10^(11)IU/mL。pAd5-SC-3M和pAd5-SC-5M感染HEK293后均能高效表达HRSV融合前构象的F蛋白并维持三聚体结构,且pAd5-SC-5M感染细胞中F蛋白表达量高于pAd5-SC-3M。结论成功在体外传代细胞中获得可高效表达A型HRSV融合前F蛋白的重组人5型腺病毒,为进一步通过动物试验评价该HRSV重组腺病毒载体疫苗奠定了基础。The purpose of the current study was to screen and construct a recombinant human type 5 adenovirus(rHADV-5)expressing human respiratory syncytial virus(HRSV)pre-conformational F protein of human respiratory syncytial virus(HRSV)and lay a foundation for further evaluation and development of RSV recombinant adenovirus vaccine.Five HRSV F protein mutants were inserted into the eukaryotic expression vector,pcDNA3.1(+).The pre-fusion F protein-monoclonal antibodies,AM14 and D25,were used to screen the expression of HRSV F protein mutant.The screened preconformational F protein mutant was inserted into the non-replicating adenovirus vector by homologous recombination,and the recombinant adenovirus was packaged and amplified in HEK293 cells.The purified recombinant adenovirus were obtained by cesium chloride density gradient centrifugation.The sandwich ELISA and Western blot methods were used to identify the expression of pre-fusion F protein.Two respiratory syncytial virus F protein sequences(SC-3M and SC-5M)that are stable in pre-fusion conformation were screened by AM14 and D25 monoclonal antibodies.The SC-3M and SC-5M coding sequences were inserted into non-replicating recombinant human type 5 adenoviruses.The titers of the two recombinant adenoviruses(pAd5-SC-3M and pAd5-SC-5M)and adenovirus vector(pAd5-vector)were 2.016×10^(10),2.52×10^(10),and 2.948×10^(11)IU/mL,respectively.pAd5-SC-3M and pAd5-SC-5M efficiently enhanced expression and maintained the conformation of the pre-fusion F protein trimer of HRSV after HEK293 infection.Expression of pre-fusion F protein in pAd5-SC-5M-infected cells was significantly higher than pAd5-SC-3M.The recombinant adenoviruses expressing pre-fusion F protein of HRSV type A were successfully obtained,and a solid foundation for further evaluation of the HRSV recombinant adenovirus vaccine through animal trials was established.

关 键 词：人5型腺病毒载体 人呼吸道合胞病毒 融合前F蛋白 HEK293细胞 

分 类 号：R373[医药卫生—病原生物学]