利用细菌人工染色体技术构建整合F基因的重组MDV疫苗株*

目的:预防马立克氏病病毒(MDV)和新城疫病毒(NDV)混合感染鸡引起的疾病,构建表达NDV F蛋白的MDV疫苗株CVI988 BAC重组载体,并包装成重组病毒,为疫苗免疫提供更多的重组疫苗选择。方法:首先利用PCR扩增带有卡那霉素(Kanamycin,Kana)抗性基因片段的F基因,采用同源重组的方法将其整合到CVI988 BAC上,进一步诱导I-SceI表达敲除Kana基因而获得重组质粒CVI988 BAC-F。通过磷酸钙法转染鸡胚成纤维细胞获得重组病毒。结果:Western blot和间接免疫荧光实验证实重组病毒能够表达F蛋白。病毒生长曲线和蚀斑大小测定结果表明,F基因的插入不影响病毒的体外增殖。结论:利用BAC技术成功构建了整合F基因的重组MDV病毒CVI988 BAC-F,为MDV重组疫苗研发,防控NDV与MDV共感染奠定了基础。

Construction of MDV Recombinant Vaccine Strain Integrated F Gene Using Bacterial Artificial Chromosome Technique

Objective: Marek’s disease virus (MDV) is an alphaherpesvirus and is classified into three serotypes: MDV serotype 1 (MDV-1) which includes the pathogenic strains and their derivatives; MDV serotype 2 (MDV-2) which consists of non-oncogenic viruses; and MDV serotype 3 (MDV-3), which is also referred to as turkey herpesvirus 1 (HVT). All three MDV serotypes have been used as vaccines. CVI988 is a cell culture passage attenuated MDV-1 virus and the gold standard among MD vaccines. CVI988 could be used as a viral vector to express exogenous gene. MDV and Newcastle disease virus (NDV) co-infection in chickens is very popular in the field, but there are few candidates of recombinant vaccines to prevent the infection of both viruses at the same time. Methods: In this study, CVI988 expressing F gene was constructed by the bacterial artificial chromosome (BAC) technology and packaged into recombinant virus. After it was amplified by PCR, the F gene together with Kana gene fragment was integrated into CVI988 BAC by homologous recombination to generate CVI988 BAC-F. Subsequently, CVI988 BAC-F was transfected into chicken embryo fibroblast (CEF) cells by calcium phosphate to rescue recombinant virus, which was confirmed by Western blotting and indirect immunofluorescence assay (IFA) assays. Results: The results of virus growth curve and plaque area measurement showed that the insertion of F gene did not affect virus proliferation in vitro. Conclusion: We successfully constructed a recombinant CVI988 BAC virus, which provides a basis for development of novel vaccines to prevent and control co-infection of NDV and MDV.