【摘要】 构建表达地方流行株轮状病毒G1型外壳蛋白VP7的复制缺陷型重组腺病毒,免疫小鼠评价其体液及细胞免疫反应效果,探讨基因工程轮状病毒疫苗的实验基础及可行性。RT-PCR扩增轮状病毒VP7基因并克隆至pshuttle-CMV穿梭质粒,pshuttle-VP7与腺病毒骨架质粒同源重组后转染293细胞包装重组腺病毒rAd-VP7。RT-PCR、western blot检测rAdVP7在体外细胞中的转录及表达。rAd-VP7经肌注或滴鼻免疫小鼠后检测其血清IgG、肠道IgA及中和抗体效价;流式、ELISPOT检测淋巴细胞亚群变化及IFN-γ分泌情况。结果显示ELISA可检测到免疫小鼠血清IgG和滴鼻组肠道IgA抗体的产生;肌注和滴鼻免疫组中和抗体平均滴度分别为228和322.5;免疫小鼠脾细胞IFN-γ的分泌增加。表达轮状病毒VP7的重组腺病毒不但能够激发体液及细胞免疫反应,滴鼻免疫途径还可诱导粘膜免疫应答。更多还原

【Abstract】 To construct a replication-defective recombinant adenovirus expressing the outer capsid protein VP7 of serotype G1 rotavirus,and evaluate its immunogenicity and efficacy in mice.VP7 gene from G1wild-type rotavirus was inserted into a shuttle plasmid(pshuttle-CMV)by double enzyme digestion.A homologous recombination was performed between pshuttle-VP7 and adenovirus skeleton genome(pAdeasy)in Ecoli BJ5183.And the linearized recombinant plasmid was transfected into Ad293 cell to produce mature adenovirus particles rAd-VP7.Female ICR mice were immunized with rAd-VP7 by intramuscular or intranasal route.The transcription and expression of VP7 could be detected by RT-PCR and Western blot assay in Vero cells.This rAd-vp7 induced antibodies production including sera IgG and intestinal secretory IgA in mice,whereas the neutralizing antibody titer was estimated at 228 and 322.5in intramuscular and intranasal group respectively.The valid IFN-γsecretions spots number of immunized group were more than those of control group,which measured using enzyme-linked immunospot(ELISPOT).The recombinant adenovirus expressing rotavirus outer capsid protein VP7 of serotype G1 induces significant humoral and cellular immune response against rotavirus.Administration by intranasal route rather than by intramuscular route produces mucosal immune response in mice.更多还原