整合NDV-F基因的重组MDV载体疫苗的构建
Construction of a recombinant MDV vector vaccine integrating the NDV-F gene
龚乐乐 1刘盼 1王乐乐 1王芮 1高美洁 1赵旭阳 1王新卫 1杜永坤 1庄国庆 1孙爱军1
作者信息
- 1. 河南农业大学动物医学院国家动物免疫学国际联合研究中心,河南郑州 450046
- 折叠
摘要
为了预防马立克氏病毒(Marek's disease virus,MDV)和新城疫病毒(Newcastle disease virus,NDV)混合感染给养禽业造成的危害,本研究以细菌人工染色体(BAC)为基因编辑平台,利用Red同源重组技术,将NDV的F基因整合到MDV双基因缺失毒株Md5 BACΔmeqΔLorf9基因组中,诱导Ⅰ-Sce Ⅰ酶表达,将筛选标记卡那霉素抗性基因敲除,构建重组活载体疫苗候选毒株 Md5 BACΔmeqΔLorf9-F.PCR以及限制性片段多态性(RFLP)分析结果显示F基因成功整合到MDV基因组且重组质粒基因组完整;将鉴定正确的重组质粒转染鸡胚成纤维细胞,1周后出现明显的细胞病变,且噬斑大小与亲本毒无明显差异;体外生长曲线结果显示,F基因的插入不影响病毒的体外复制能力.结果表明,本研究正确构建了表达NDV保护性抗原F基因的重组MDV,为研制ND重组MDV活载体疫苗奠定了基础,对NDV和MDV混合感染的防控具有重要意义.
Abstract
In order to prevent co-infection of Marek's disease virus(MDV)and Newcastle disease virus(NDV)in clinic,this study used bacterial artificial chromosome(BAC)as the gene editing platform to integrate the F gene of NDV into the genome of MDV double-gene deletion strain Md5 BAC ΔmeqΔLorf9 through two-step Red-mediated homologous recombination technology.The ex-pression of Ⅰ-SceⅠ enzyme was induced to knocked out the kanamycin resistance gene,and the recombinant live vector vaccine candidate strain Md5 BACΔmeqΔLorf9-F was constructed.PCR and restriction fragment polymorphism(RFLP)assays confirmed the integrity and correctness of MDV BAC clones containing the F gene.Then,the recombinant plasmid was transfected into chicken embryo fibroblasts to obtain recombinant virus,which was identified by indirect immuno-fluorescence assay.The results of plaque size determination and in vitro growth curve showed that the insertion of F gene did not affect viral replication in vitro.Meanwhile,IFA test confirmed the expression of F protein.Overall,these results indicate that we successfully construct recombinant MDV expressing NDV protective antigen F gene,which provides basis for the prevention and con-trol of NDV and MDV co-infection.
关键词
细菌人工染色体/马立克氏病病毒/新城疫病毒/活载体疫苗Key words
bacterial artificial chromosome/Marek's disease virus/Newcastle disease virus/live vec-tor vaccines引用本文复制引用
基金项目
国家自然科学基金资助项目(U21A20260)
河南省高等学校重点科研资助项目(22A230011)
河南农业大学高层次"拔尖人才"资助项目(30500690)
出版年
2024
NETL
NSTL
万方数据