含LoxP位点和EGFP的鸭瘟病毒TK基因缺失转移载体的构建  

Construction of TK gene-deleted DPV transfer vector containing Loxp and EGFP

作　　者：潘华奇[1] 刘磊[1] 曹瑞兵[2] 魏凡华[3] 袁立科[1] 李娟[1] 孙海亮[1] 潘光炎[1] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]南京农业大学农业部动物疫病诊断与免疫重点开放实验室,江苏南京210095 [3]宁夏大学农学院动物科学系,宁夏银川750021

出　　处：《甘肃农业大学学报》2008年第2期8-12,共5页Journal of Gansu Agricultural University

摘　　要：提取鸭瘟病毒(DPV)疫苗株基因组DNA为PCR扩增模板,并根据GenBank已发表的DPV UL区UL24、UL23、UL22基因序列,选择保守序列设计了两对引物,分别扩增出位于UL23(TK)两侧的可用于同源重组的左臂片段(L)和右臂片段(R),L包括部分UL24、部分TK,R包括部分TK及部分UL22,L和R的拼接片段中TK基因内部缺失468个核苷酸.将L片段和R片段克隆于pBluescript M13-载体上,获得了pSKLR;再将含2个LoxP位点的表达绿色荧光蛋白的基因表达盒插入pSKLR的L片段和R片段之间构成转移载体pSKLR-EGEP-LoxP.大量提取pSKLR-EGEP-LoxP质粒,用脂质体转染鸭胚成纤维细胞,24 h后蛋白被正确表达,表明含LoxP位点的表达EGFP的鸭瘟病毒TK基因缺失的重组转移载体被成功构建.DPV vaccine strain genome DNA was extracted and two pairs of primers were synthesized according DPV vaccine strain nucleotide sequence published on GenBank,one pairs of the primers was used to amplify the left recombinant fragment（L） of 911 bp containing the partial UL 24 and partial TK, the other to amplify the right recombinant fragment （R） of 628 bp containing the partial TK and partial UL22. The left and right fragments were cloned in to pBluescript-M13 to obtain the transfer vector pSKLR. EGFP gene containing two Loxps from PRV pSKLR-EGEP-LoxP was inserted into pSKLR between L and R,and then transfer vector DPV pSKLR-EGEP-LoxP was obtained. The transfer vector DPV pSKLR-EGEP-LoxP was transiently transfected into DEF,and then the green fluorescence was observed. The results showed that EGFP gene could be expressed correctly.

关 键 词：鸭瘟病毒 EGFP Cre/LoxP TK基因 转移载体 

分 类 号：S858.32[农业科学—临床兽医学] Q782[农业科学—兽医学]