重组腺病毒5型-诺如病毒GⅡ.4型-VP1病毒样颗粒的表达及鉴定  

Expression and identification of recombinant adenovirus type 5⁃Norovirus GⅡ.4⁃VP1 virus⁃like particles

作　　者：汪梦俊 孙楠 裴捷 刘睿伦 邹文琪 熊宇 王文辉 于代冠 申硕 WANG Mengjun;SUN Nan;PEI Jie;LIU Ruilun;ZOU Wenqi;XIONG Yu;WANG Wenhui;YU Daiguan;SHEN Shuo(Viral Vaccine Research Laboratory,Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,Hubei Province,China)

机构地区：[1]武汉生物制品研究所有限责任公司病毒性疫苗研究一室,湖北武汉430207

出　　处：《中国生物制品学杂志》2024年第2期138-142,共5页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2020YFC0842100,2020YFC0860600)。

摘　　要：目的 采用293T细胞表达重组腺病毒5型(recombinant adenovirus 5,rAd5)-诺如病毒(Norovirus,NoV)GⅡ.4型-VP1病毒样颗粒(virus-like particle,VLP),并进行鉴定。方法 将重组腺病毒质粒pAd5-eGFP及pAd5-NoV-GⅡ.4-VP1分别转染293T细胞,拯救获得重组腺病毒rAd5-eGFP和rAd5-NoV-GⅡ.4-VP1。rAd5-eGFP在293T细胞上连续传代,验证其载体功能。将rAd5-NoV-GⅡ.4-VP1在293T细胞上进行传代,NoV-GⅡ.4-VP1经表达及纯化后,自组装形成NoV-GⅡ.4-VLP,并进行Western blot、ELISA检测及电镜观察。结果 镜下观察各代rAd5-eGFP均可见绿色荧光,且亮度随代次增加而增强。各代rAd5-NoV-GⅡ.4-VP1收获液中均可见NoV-GⅡ.4-VP1蛋白表达,相对分子质量约58 900。NoV-GⅡ.4-VLP可与兔抗NoV-GⅡ.4-VP1血清发生特异性结合,具有与天然的NoV病毒颗粒相似构象,可有效识别志愿者唾液样本中的NoV受体;镜下观察,其形态完整,呈球状,直径为43.5~58.3 nm,颗粒表面有少数突起,可能为VP1自组装时暴露于颗粒表面的P结构域。结论 表达的重组腺病毒NoV-GⅡ.4-VLP具有完整的VLP结构和良好的特异性,有望用于NoV腺病毒载体疫苗的相关研究。Objective To express and identify recombinant adenovirus type 5-Norovirus(NoV)GⅡ.4-VP1 virus-like particles(VLPs)in 293T cells.Methods The recombinant adenovirus plasmids pAd5-eGFP and pAd5-NoV-GⅡ.4-VP1 were transfected into 293T cells respectively,and the recombinant adenovirus rAd5-eGFP and rAd5-NoV-GⅡ.4-VP1 were rescued.The rAd5-eGFP was subcultured in 293T cells to verify the function of the vector.The rAd5-NoV-GⅡ.4-VP1 was subcultured in 293T cells,expressed and purified,and then NoV-GⅡ.4-VLP was formed by self-assembly,which was detected by Western blot,ELISA and observed by transmission electron microscope.Results The green fluorescence of the recombinant adenovirus rAd5-eGFP of various generations was observed under microscope,and the brightness increased with the increase of generations.NoV-GⅡ.4-VP1 protein was expressed in the harvested solution of recombinant adenovirus rAd5-NoV-GⅡ.4-VP1 of various generations,with a relative molecular mass of about 58900.NoV-GⅡ.4-VLP showed specific binding to the rabbit anti-NoV-G II.4-VP1 serum;it had similar conformation to natural NoV virus particles and can effectively identify NoV receptors in volunteer saliva samples;microscopic observation showed that the morphology was complete and spherical,with a diameter of 43.558.3 nm,while there were a few protrusions on the surface of the particles,which might be the P domain exposedon the particle surface during self-assembly of VP1.Conclusion The expressed recombinant adenovirus NoV-G II.4-VLP has complete VLP structure and good specificity,and is expected to be used in the related research of NoV adenovirus vector vaccine.

关 键 词：腺病毒 诺如病毒 VP1 病毒样颗粒 腺病毒载体疫苗 

分 类 号：R183.3[医药卫生—流行病学]