【摘要】 提取伪狂犬病毒(PRV)Bartha-K61株基因组DNA为PCR扩增模板,并根据GenBank已发表的PRVKa plan株UL区UL25、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(含部分TK基因)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UL25、全部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270个核苷酸。将L片段和R片段克隆于pBluescriptM13-载体上,获得pSKLR;再将绿色荧光蛋白载体pEGFP-C1上含GFP及其多克隆位点的完整的基因表达盒插入pSKLR的L片段和R片段之间构成转移载体pSKLRG。提取pSKLRG质粒,经单酶切线性化后用脂质体与PRVBartha-K61株共转染143TK-细胞,在细胞培养液中有5 溴脱氧尿嘧啶(BrdU)存在的条件下筛选出表达绿色荧光蛋白的重组PRVBartha-K61毒株:PRVrBGFP。更多还原

【Abstract】 PRV Barthak61 strain genome DNA was extracted and two pairs of primers were synthesized according PRV kaplan strain nucleotide sequence published on genbank,one pairs of the primers used to amplify the left recombinant fragment (L) of 1167 bp containing the partial UL25,the whole UL24 and partial TK,the other to amplity the right recombinant fragment (R) of 2083 bp containing the partial TK and partial UL22.The left and right fragments were cloned into pBluescript M13 to obtain the transfer vector pSKLR.EGFP gene from PEGFPC1 was inserted into PSKLR between L and R to obtain transfer vector pSKLRG,and then pSKLRG was transfected into 143 TKcells with Bartha strain virus,the transfected cells could emit green fluorescence and one recombinant rBGFP was obtain with screen tests using BUDR.更多还原