表达PEDV中国变异株S蛋白复制型重组人腺病毒4型的构建及其免疫原性研究

摘要

In order to construct a replicative recombinant human adenovirus serotype 4 (HAdV-4) expressing the S protein of PEDV and evaluate the immunogenicity of the recombinant virus.The N-terminal of S gene (1 bp-1 140 bp) in PEDV Chinese variant strain was cloned into shuttle vector pAd4FAST-Shuttle.The constructive recombinant plasmid pAd4FAST-Shuttle-S was linearized,and transformed into BJ5183 host harboring HAdV-4 backbone plasmid for homologous recombination to get the infectious clone of HAdV-4 containing the PEDV S gene.The linearized infectious clone DNA was transfected into HEK293 cells to rescue the recombinant virus rAd4△E3-S,and the immunogenicity of rAd4△E3-S was evaluated with mouse model.Antibody responses against PEDV S protein were determined by indirect ELISA,and the T lymphocyte responses were evaluated by lymphocyte proliferation assay and flow cytometry.The results showed that the rAd4△E3-S could induce specific humoral and cellular immune responses against PEDV spntein and the results might lay a foundation for clinical evaluationin of the Ad4△E3-S with swine model.%为构建表达猪流行性腹泻病毒(PEDV)S蛋白复制型重组人腺病毒4型(HAdV-4)及评价其免疫原性,本研究利用RT-PCR扩增得到PEDV中国变异株纤突(S)基因的N-末端区域(1 bp～1 140 bp),构建重组质粒pAd4FAST-Shuttle-S,重组质粒经线性化和同源重组,得到含有PEDV S基因的重组HAdV-4感染性克隆,纯化的感染性克隆DNA经Pac Ⅰ线性化后转染HEK293细胞,拯救得到表达PEDV变异株S蛋白的重组病毒rAd4△E3-S.将该重组病毒免疫小鼠,间接ELISA测定其血清中抗S蛋白抗体水平,利用体外淋巴细胞转化试验和流式细胞技术检测免疫小鼠T淋巴细胞反应.结果显示,重组病毒能够表达PEDV S蛋白,并且能刺激小鼠产生特异性体液和细胞免疫应答.本实验结果为进一步研究重组病毒在猪体上的免疫反应奠定基础,也为PEDV活载体疫苗的研发提供实验依据.