摘要
用限制性核酸内切酶Sall酶切已缺失gⅠ (gE)和部分gp63 ( gⅠ )的重组质粒PPB7- 1DNA后 ,回收大小约 6 0kb片段 ,经T4 DNA连接酶连接后转化入大肠杆菌DH5α株 ,得到含 gp63片段的转移载体质粒PP63。再以SV4 0早期启动子控制的大肠杆菌 β 半乳糖苷酶 (LacZ)基因作为报告基因 ,将其插入PP63中 gp63基因起始密码子ATG下游的StuⅠ位点 ,在选择培养基中筛选出表达 β 半乳糖苷酶的转化子 ,构建了以gp63为同源序列含LacZ报告基因的转移载体质粒 ,命名为PP63LacZ。在该转移载体质粒中 ,LacZ基因的两端分别与 0 3kb和 0 5kb的PRVgp63同源序列相连 ,经酶切、核酸分子杂交技术鉴定 。
WT5BZ]By means of restriction enzyme digestion,ligation and insertion the transfer vector plasmid PP63LacZ of Pseudorabies virus Fa strain was obtained based on plasmid PPB7 1 deleted gI(gE) and part of gp63(gI).In this paper,18 recombinant plasmids were obtained all contained gp63 gene.These plasmids were named PP63.After digestion with EcoR Ⅰ and Hind Ⅲ,the reporting gene LacZ from plasmid PCMV β was inserted into the StuⅠ site downstream of initination codon ATG of gp63 gene,the transfer vector plasmid PP63LacZ was constructed.Assaied using the mothods of Dot blot,southern blot acid hybridization,digestion with different restriction enzymes,the results suggested that the construction of PP63LacZ was successful. [WT5HZ]
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2001年第4期338-344,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金
国家"九五"科技 (攻关 )资助项目
