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

作　　者：龚乐乐 刘盼 王乐乐[1] 王芮 高美洁 赵旭阳 王新卫[1] 杜永坤 庄国庆 孙爱军 GONG Lele;LIU Pan;WANG Lele;WANG Rui;GAO Meijie;ZHAO Xuyang;WANG Xinwei;DU Yongkun;ZHUANG Guoqing;SUN Aijun(International Joint Research Center of National Animal Immunology,College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[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

出　　处：《中国兽医学报》2024年第1期52-58,共7页Chinese Journal of Veterinary Science

基　　金：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)。

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-SceⅠ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。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 expression 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 immunofluorescence 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 control 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 

分 类 号：S852.65[农业科学—基础兽医学]