表达戊型肝炎病毒ORF2蛋白的重组腺病毒构建及其经粘膜途径免疫小鼠

Construction of a Recombinant Human Adenovirus Expressing the ORF2 Antigen of HEV and Immunization of Mice by Mucosal System

目的构建可表达戊型肝炎病毒(hepatitisEvirus,HEV)ORF2蛋白(112-660aa)的重组腺病毒Adasy-EORF2,探讨其经鼻腔和腹腔免疫小鼠后,诱导机体产生特异性体液免疫和粘膜免疫应答的能力,为研制戊型肝炎疫苗探索新的途径。方法经PCR扩增编码ORF2蛋白(112-660aa)的基因片段,将其连接于重组腺病毒Adasy系统的穿梭质粒pTrack-CMV上,与腺病毒基因组质粒pAdEasy-1共转化宿主菌BJ5183,经细菌内同源重组获得重组腺病毒基因组质粒,转染293细胞以产生、扩增重组腺病毒。将重组腺病毒以107pfu的剂量经鼻腔和腹腔免疫小鼠,通过ELISA检测血液IgG和粘膜IgA的应答情况。结果重组腺病毒经鼻腔免疫小鼠后可刺激机体产生特异性IgA、IgG免疫应答,IgG最高滴度达1∶1000。经腹腔免疫可刺激小鼠产生IgG免疫应答,最高滴度可达1∶10000,未检测到明显的IgA应答。结论表达HEVORF2蛋白的重组腺病毒能刺激机体产生有效的免疫应答,具有成为新型戊型肝炎疫苗的潜力。

Objective To construct a replication-defective recombinant adenovirus expressing the ORF2(112-660aa)antigen of hepatitis E virus(HEV)and evaluate its immunization effect in BALB/c mice by mucosal inoculation.Methods The HEV ORF2gene encoding for112-660aa was amplified from plasmid pUC-HEV and inserted into the transfer vector pTrack-CMV.The recombinant plasmid and adenoviral backbone plasmid pAdEasy-1were co-transformed into E.coli strain BJ5183.Taking the advantage of the high efficient homologous recombination machinery presented in bacteria,the recombinant adenovirus backbone plasmid was generated in BJ5183,and then was transfected into293cells.Recombinant Adenoviruses were propagated in293cells with high titers.8-week-old BALB/c mice were inoculated intraperitoneally and intranasally with10 7 pfu recombinant adenovirus each on weeks0,3,5,7,10.Results Both groups of mice induced humoral IgG immune response with the highest titers1∶1000and1∶10000each.Only the group inoculated intranasally could induce mucosal IgA immune response.Conclusions The adenoviral recombinant can stimulate specific humoral and mucosal immune response in mice and is potentialy to be used as a candidate vaccine for the treatment of HEV infection.