摘要
为开展鸭瘟病毒-鸭坦布苏病毒二联苗的研究,将密码子优化的鸭坦布苏病毒(DTMUV)E基因插入转移载体p EP-BGH-end构建了p EP-BGH-E重组表达质粒。在鸭瘟病毒疫苗株细菌人工染色体(p DEVvac)的基础上,首先通过Red E/T两步重组法构建了EF1启动子替换GFP基因CMV启动子的p DEV-EF1突变体克隆,并将p EP-BGH-E质粒上的重组表达框p CMV-E-BGH-p A再次通过两步重组克隆至p DEV-EF1突变体的US7和US8基因之间,构建了携带有外源基因DTMUV E的突变体克隆p DEV-E。用磷酸钙法转染鸡胚成纤维细胞(CEFs)获得了重组病毒r DEV-E。病毒蚀斑大小测定结果显示,r DEV-E在CEFs上的蚀斑面积较亲本株相比稍有减少。Western blotting分析表明,外源蛋白E在病毒感染的CEFs细胞中成功表达。
To develop duck enteritis virus（ DEV） and duck Tembusu virus（ DTMUV） bivalent vaccine,the recombinant plasmid p EP-BGH-E（ containing expression cassette p CMV-E-BGH-p A） was constructed by inserting codonoptimized DTMUV E gene into the expression vector p EP-BGH-end. The BAC clone of p DEV-E was generated by twice Red / ET recombination in E. coli based on the bacterial artificial chromosome（ BAC） clone p DEV-vac which carried whole DEV genome. Firstly,the BAC clone p DEV-EF1 was constructed by replaced CMV promoter of GFP gene with EF1 promoter on p DEV-vac; then p DEV-E was constructed by inserting p CMV-E-BGH-p A between DEV US7 and US8 gene on p DEV-EF1. The recombinant virus r DEV-E was rescued from chicken embryo fibroblasts（ CEFs） transfected with p DEV-E by calcium phosphate precipitation. The plaque size of r DEV-E was slightly decreased compared with the parental virus r DEV-BAC. Western blotting analysis showed that DTMUV E protein wasexpressed in r DEV-E virus-infected CEFs.
出处
《浙江农业学报》
CSCD
北大核心
2015年第11期1889-1895,共7页
Acta Agriculturae Zhejiangensis
基金
浙江省科技计划项目优先主题重点国际科技合作研究项目(2011C14011)
浙江省重点科技创新团队项目(2010R50027)
浙江省自然科学基金项目(LY15C180002
LY13C180002)
关键词
鸭瘟病毒
鸭坦布苏病毒
E蛋白
细菌人工染色体
重组病毒
duck enteritis virus
duck Tembusu virus
E protein
bacterial artificial chromosome
recombinant virus
