摘要
目的本研究目的为构建重组穿梭载体,拯救带有增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)重组病毒,为重组二联苗的研制奠定基础。方法选择已经证实的US区域的US2-SORF3、US7-US8和UL区域中的UL15-UL18的非编码区作为插入位点构建穿梭载体P-US2-SORF3-EGFP,P-UL15-UL18-EGFP,P-US7-US8-EGFP。在已感染疫苗毒的鸡胚成纤维细胞(chicken embryo fibroblasts,CEFs)中转染穿梭载体,经细胞同源重组,收集细胞上清,进行噬斑筛选及纯化。结果成功构建了三个带有EGFP标签的穿梭载体,p Blusecript II SK构建的穿梭载体未成功筛选到重组噬斑而用p UC18构建的穿梭载体成功筛选到重组噬斑。结论拯救鸭瘟重组毒的研究中,p Blusecript II SK载体可能不是最佳的适用载体,也可能是插入位点为病毒复制的必须区。
Objective The aim of this study was to construct shuttle vectors and rescue the recombinant viruses with EGFP, which provided basic research for bivalent live vaccine of DPV. Method We selected the proven insertion sites, including US region of US2-SORF3, UST-US8 and UL region of UL15-UL18 unconding regions of DPV to construct shuttle vectors P-US2-SORF3-EGFP, P-UL15-UL18-EGFP and P-UST-US8-EGFP. Shuttle vectors were used to transfect the CEFs infected with vaccine strain by using liposome 2000. The recombinant viruses were rescued by homologous recombination. Then collected the cells supernant and purified plaques. Result We successfully constructed three shuttle vectors with EGFP. We failed to rescue any recombinant virus plaques by using pBlusecript II SK vector. However, the recombinant virus plaques were rescued with pUC18 vector. Conclusion It was suggested that pBluseeript II SK vector might be unsuitable for rescuing recombinant viruses, and the insertion site might also affect the virus replication.
出处
《实验动物科学》
2017年第3期16-19,23,共5页
Laboratory Animal Science
基金
黑龙江省地方科技攻关计划课题(No.2014QA3BN002)
黑龙江省自然科学基金重点项目(No.ZD2016006)
关键词
鸭瘟病毒
穿梭载体
噬斑纯化
duck enteritis virus
shuttle vector
plaque purification
