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发明公开
CN107475296A 一种表达鸡4型腺病毒fiber2基因的重组鸡痘病毒转移载体及其构建方法和应用 有权

一种表达鸡4型腺病毒fiber2基因的重组鸡痘病毒转移载体及其构建方法和应用
摘要：
本发明属于生物技术领域，主要涉及一种表达鸡4型腺病毒(FADV4)fiber2基因的重组鸡痘病毒转移载体及其构建方法和应用。所述载体以pMD19T-Simple载体为基础，TA克隆位点插入FADV4fiber2基因、lacz基因，以及用于同源重组的鸡痘病毒的基因组复制非必须片段LTYB和RTYB。所述方法包括步骤如下：质粒pMD-TYB；构建质粒pMD22；构建质粒pMD22-lacz；构建中间载体pMD22-TYB-lacz；扩增FADV4 fiber2基因；构建重组鸡痘病毒转移载体pMD22-TYB-lacz-F4；将转移载体pMD22-TYB-lacz-F4与鸡痘病毒共转染鸡胚成纤维细胞，鉴定选出阳性，继续传代培养；鉴定重组鸡痘病毒的表达效果。本发明构建的重组鸡痘病毒转移载体为研制高效的表达鸡4型腺病毒的重组鸡痘病毒基因工程活载体疫苗奠定基础。
摘要（英）：

The invention belongs to the technical field of biology, and mainly relates to a recombinant fowlpox virus transfer vector expressing a fowl adenovirus serotype-4 (FADV4) fiber2 gene, a constructing method thereof and applications of the transfer vector. A pMD19T-Simple vector is adopted as a base of the transfer vector. The FADV4 fiber2 gene, a lacz gene, and fowlpox virus genome replicated non-essential fragments which are LTYB and RTYB used for homologous recombination are inserted in a TA cloning site. The method includes constructing a plasmid pMD-TYB; constructing a plasmid pMD22; constructing a plasmid pMD22-lacz; constructing an intermediate vector pMD22-TYB-lacz; amplifying the FADV4 fiber2 gene; constructing a recombinant fowlpox virus transfer vector pMD22-TYB-lacz-F4; subjecting a chicken embryo Fibroblast to cotransfection with the transfer vector pMD22-TYB-lacz-F4 and a fowlpox virus; performing identification to select a positive product; performing subculture continuously; and identifying expression effects of the recombinant fowlpox virus. The recombinant fowlpox virus transfer vector constructed by the method lays a foundation for development of an efficient recombinant fowlpox virus genetic engineering living-vector vaccine expressing the fowl adenovirus serotype-4.

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