新城疫病毒F48E8株核衣壳蛋白基因在鸡痘病毒中的表达及其部分序列分析  

Expression of the Nucleocapsid Gene of Newcastle Disease Virus Strain F48E8 in Fowlpox Virus and Analysis of the Nucleocapsid Gene Partial Sequence

作　　者：姜焱[1] 刘伟忠[1] 吴艳涛[1] 彭大新[1] 张如宽[1] 刘秀梵[1] 

机构地区：[1]扬州大学农学院动物医学系,扬州225009

出　　处：《农业生物技术学报》1999年第3期231-235,共5页Journal of Agricultural Biotechnology

基　　金：江苏省自然科学基金

摘　　要：通过反转录聚合酶链反应（ＲＴＰＣＲ） 扩增了新城疫病毒（ＮＤＶ）Ｆ４８Ｅ８ 株核衣壳蛋白（ＮＰ） 基因，并克隆到ｐＵＣ１８ 中，得阳性克隆ｐＵＣＮＰ。应用分子克隆技术，将ＮＰ基因导入鸡痘病毒（ＦＰＶ） 转移载体ｐＦＧ１１７５１ 中Ｐ７５ 启动子的下游，得到含ＮＤＶ ＮＰ基因的质粒ｐＦＧＮＰ１１７５１ 。利用脂质体转染技术，将该质粒转染ＦＰＶ 疫苗株２８２Ｅ４ 感染３ ～４ ｈ 的鸡胚成纤维细胞，采用蓝斑筛选方法纯化多次，得到稳定的重组ＦＰＶ。用狄高辛标记的探针进行斑点杂交实验，结果表明ｐＦＧＮＰ１１７５１ 已与ＦＰＶ 发生同源重组；用抗ＮＤＶ 的ＳＰＦ 鸡的阳性血清进行间接免疫荧光实验，证实重组ＦＰＶ 在感染细胞中表达了Ｆ４８Ｅ８ 株ＮＰ蛋白。该基因两端部分序列与已发表的Ｄ２６ 株ＮＰ 基因对应区域的核苷酸同源性为８９１ ％ ，推断的氨基酸同源性则为９３１ ％ 。For the construction of transfer vector pFGNP1175 1 ,the gene encoding Nucleocapsid Protein of newcastle disease virus strain F48E8 was removed from plasmid pUCNP,and inserted into \%Hin\%dIII site of pFG1175 1, downstream of p7 5 promoter. Chicken embryo fibroblast (CEF) cell cultures which were infected with FPV 282E4 for 3～4 h were transfected with the complex of pFGNP1175 1 plasmid and DOSPER liposomal. Recombinant FPV was selected and purified many times in CEF cell cultures overlaid with agar containing X gal through blue plaques. The expression of NDV F48E8 NP gene in FPV was proved by indirect immunofluorescence assay with antiserum of NDV. The NP gene of NDV F48E8 was partial sequenced. As compared with the NP gene encoding region of NDV avirulent D26,the homology of nucleotide was 89 1 % and the deduced amino acid homology was 93 1 %.

关 键 词：鸡痘病毒 新城疫病毒 核衣壳蛋白 部分序列分析 

分 类 号：S858.312.6[农业科学—临床兽医学]