腺病毒载体表达轮状病毒G1型外壳蛋白VP7及其诱导的免疫应答研究

发布时间：2018-05-04 00:08

本文选题：轮状病毒 + VP　；参考：《现代免疫学》2015年02期

【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。
[Abstract]:A replication-deficient recombinant adenovirus expressing G1 coat protein VP7 of rotavirus was constructed and immunized mice to evaluate its humoral and cellular immune response. To investigate the Experimental basis and Feasibility of genetically engineered Rotavirus Vaccine. RT-PCR amplification of rotavirus VP7 gene and cloning into pshuttle-CMV shuttle plasmid pshuttle-VP7 homologous recombination with adenovirus skeleton plasmid, then transfection into 293 cell packaging recombinant adenovirus rAd-VP7.RT-PCRrwestern blot detection The transcription and expression of rAdVP7 in vitro. RAd-VP7 was immunized by intramuscular or nasal drip to determine the titer of serum IgG, intestinal IgA and neutralizing antibody. Lymphocyte subsets and IFN- 纬 secretion were detected by flow cytometry. The results showed that ELISA could detect the production of intestinal IgA antibody in serum IgG and nasal drip group, the average titers of neutralizing antibody were 228 and 322.5 in intramuscular and nasal immunization groups, respectively, and the secretion of IFN- 纬 in spleen cells of immunized mice was increased. Recombinant adenovirus expressing rotavirus VP7 can not only stimulate humoral and cellular immune responses, but also induce mucosal immune responses.
【作者单位】：昆明学院医学院;中国医学科学院医学生物学研究所北京协和医学院云南省重大传染病疫苗研发重点实验室;昆明医科大学;
【基金】：国家科技重大专项资助(20132X10004002-003-05) 云南省教育厅科学研究基金(2013Y256)
【分类号】：R392.11

【参考文献】