表达新城疫病毒HN基因的重组火鸡疱疹病毒的构建

Construction of a Recombinant Herpesvirus of Turkey Expressingthe Haemagglutinin Neuraminidase Gene of Newcastle Disease Virus

应用聚合酶链反应技术，用设计带有限制性酶切位点的引物，特异地扩增从起始密码子开始的１ ．８ ｋｂ 新城疫病毒（ Ｎ Ｄ Ｖ） 血凝素神经氨酸酶（ Ｈ Ｎ） 基因开放式阅读框，然后插入ｐ Ｔ Ｋ２ Ｂ 的 Ｎｈｅ Ⅰ位点，构建了含 Ｎ Ｄ Ｖ Ｈ Ｎ 基因的插入载体ｐ Ｔ Ｋ Ｈ Ｎ１ 和ｐ Ｔ Ｋ Ｈ Ｎ２ ，再与感染火鸡疱疹病毒（ Ｈ Ｖ Ｔ） 细胞的总 Ｄ Ｎ Ａ 共转染鸡胚成纤维细胞（ Ｃ Ｅ Ｆ） ，经有限稀释法和 Ｄｏｔｂｌｏｔ 筛选，得到含有 Ｈ Ｎ 基因的重组体ｒ Ｈ Ｖ Ｔ１ 和ｒ Ｈ Ｖ Ｔ２ 。经组织培养传代和 Ｗｅｓｔｅｒｎ ｂｌｏｔ 分析，表明重组体在感染细胞中表达了 Ｈ Ｎ 蛋白。重组体在 Ｃ Ｅ Ｆ 上的生长特性与亲本病毒相同，且在连续传代过程中保持稳定。为国内新城疫基因工程疫苗研制奠定了基础。

A 1.8 kb fragment of Newcastle disease virus (NDV) haemagglutinin neuraminidase (HN) gene was amplified by polymerase chain reaction (PCR) with primers containing enzyme cleavage sites, and inserted into a nonessential gene of herpesvirus of turkey (HVT). Recombinant viruses were screened by Dot blot hybridization with digoxigenin labelled probe and purified by plaque formation technique. The expression of NDV HN gene can be detected by Western blotting in CEF cells infected with recombinant virus. Recombinant HVT strains were replicated stably in cell cultures.