脑心肌炎病毒VP1基因重组腺病毒的构建及对小鼠免疫效果的研究

发布时间：2018-06-04 00:43

本文选题：脑心肌炎病毒 + 结构蛋白VP　；参考：《中国兽医科学》2017年03期

【摘要】：为构建脑心肌炎病毒(EM CV)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p Yr-adshuttle-4连接构建p Yr-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p Ad-VP1-EG FP重组腺病毒载体,将此载体于H EK 293细胞中包装最终成功获得r Ad-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r Ad-VP1-EGFP,测得其TCID50为10-8.5/m L;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。
[Abstract]:To construct recombinant adenovirus of EM CV)VP1 gene of encephalitis virus, and to provide experimental basis for the development of EMCV vaccine. The EMCV VP1 gene was amplified and ligated with the vector p Yr-adshuttle-4 to construct the p Yr-ads-4-VP1 shuttle vector. The recombinant adenovirus vector of p Ad-VP1-EG FP was obtained by in vitro recombination of the shuttle plasmid with the adenovirus vector. The recombinant adenovirus was successfully packaged in HEK293 cells to obtain r Ad-VP1-EGFP recombinant adenovirus, and its TCID50 was determined. The recombinant adenovirus was infected with the recombinant adenovirus at gene level and protein level, and VP1 was detected at the gene level and protein level. Finally, the mice were immunized with recombinant adenovirus by EMCV attack test to verify its immune effect. The results showed that the recombinant adenovirus r Ad-VP1-EGFP of EMCV VP1 gene was successfully constructed, and its TCID50 was 10-8.5 / mL. The expression of VP1 gene could be detected by gene level and protein level after infection. The recombinant adenovirus had a better immune effect on mice, and the protective rate after secondary immunization was significantly higher than that of single immunization. The results showed that the recombinant adenovirus of EMCV gene was successfully prepared in this experiment, and it has a good immune effect on mice, which can lay a foundation for the further development of EMCV gene engineering vaccine.
【作者单位】：西北民族大学生物工程与技术国家民委重点实验室;西北民族大学生命科学与工程学院;甘肃省军区甘南州军分区;甘肃省动物细胞工程技术研究中心;
【基金】：国家自然科学基金项目(31460665) 教育部“长江学者和创新团队发展计划”项目(IRT13091) 中央高校基本科研业务费专项资金项目(31920150030,3192015005,Zy P2015005)
【分类号】：S852.65

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