中文摘要：

为构建脑心肌炎病毒（EMCV）VP1基因的重组腺病毒,以期为EMCV疫苗研制提供试验依据。扩增EMCV VP1基因并与载体pYr-adshuttle-4连接构建pYr-ads-4-VP1穿梭载体;穿梭质粒通过体外重组与腺病毒载体进行重组,获得pAd-VP1-EGFP重组腺病毒载体,将此载体于HEK293细胞中包装最终成功获得rAd-VP1-EGFP重组腺病毒,并测定其TCID50;构建的重组腺病毒感染细胞后在基因水平、蛋白水平对VP1进行检测;最后通过对小鼠免疫重组腺病毒后进行EMCV攻毒试验,验证其免疫效果。结果显示,成功构建了EMCV VP1基因重组腺病毒rAd-VP1-EGFP,测得其TCID50为10-8.5/mL;感染细胞后基因水平及蛋白水平均可检测到VP1基因的表达;重组腺病毒免疫小鼠后的存活情况表明,重组腺病毒对小鼠有较好的免疫效果,且二次免疫后保护率显著高于单次免疫。结果表明,本试验成功制备了EMCV基因重组腺病毒,且对小鼠具有较好的免疫效果,可为进一步开发EMCV基因工程疫苗奠定基础。

英文摘要：

By construction of recombinant adenovirus with encephalomyocarditis virus（EMCV） VP1 gene,the study aims to offer experimental basis for EMCV vaccine development.EMCV VP1 gene was amplified,and linked with pYr-adshuttle-4 to construct the shuttle vector pYr-ads-4-VP1.The shuttle vector with adenovirus vector by in vitro recombination was constructed,designed as recombinant adenovirus vector p Ad-VP1-EGFP,which was finally packaged into HEK293 cells successfully to obtain recombinant adenovirus rAd-VP1-EGFP.Cells were infected with this recombinant adenovirus,then VP1 in the m RNA andprotein levels.Finally,mice were immunized with r Ad-VP1-EGFP,and its immune effects were verified.The results showed that the recombinant adenovirus rAd-VP1-EGFP was successful,and TCID50 was 10-8.5/mL.VP1 was expressed in gene both the mRNA and protein levels after infected with recombinant adenovirus.The survival rate indicated that rAd-VP1-EGFP had a good immune effect on mice,significantly higher than single immunination.The above-results laid the foundation for further EMCV genetic engineering vaccine development.