摘要
利用昆虫杆状病毒表达系统表达水貂肠炎病毒(mink enteritis virus,MEV)VP2基因,表达的蛋白能够形成病毒样粒子,具有反应原性,为研究MEV新型疫苗奠定基础。采用PCR方法扩增MEVVP2基因,将PCR产物连接到pMD18-T载体,目的基因定向克隆到pFast-BacⅠ载体中,构建重组转座载体后转化DH10Bac感受态细胞,获得重组Bacmid质粒后转染Sf9昆虫细胞,传毒3代,对表达蛋白进行Western blotting鉴定。结果成功克隆MEVVP2基因。Western blotting结果证实,表达蛋白能够被MEV单克隆抗体识别。在Bac-To-Bac杆状病毒表达系统中成功的表达了MEV VP2蛋白,目的蛋白具有较好的反应原性。
To obtain expression of VP2 gene of MEV in Bac-To-Bac expression system and develop MEV subunit vaccine.MEV VP2 gene was amplified by PCR method and then cloned into pMD18-T vetcor.The VP2 gene was inserted into pFast-BacⅠ vector,then transferred the recombinant plasmid pFast-BacⅠ-VP2 into DH10 Bac to get recombinant Bacmid.Subsequently,the recombinant bacmid was transfected into Sf9 cells.The expressed recombinant protein was identified by Western blotting after three cell passages.VP2 gene of MEV was successfully cloned.The obtained recombinant VP2 protein reacted with MEV monoclonal antibody was identified by Western blotting.The VP2 gene of MEV is successfully expressed in Bac-To-Bac expression system and shows well reactivity with MEV monoclonal antibody.
出处
《中国畜牧兽医》
CAS
北大核心
2011年第9期97-100,共4页
China Animal Husbandry & Veterinary Medicine
基金
科技部科研院所社会公益研究专项"野生动物重要传染病生态学发生
监测和控制技术"(2005DIB4J048)
