含PRRS病毒ORF5的伪狂犬病病毒TK基因缺失转移载体的构建

Construction of the Thymidine Kinase Gene Deleted Pseudorabies Virus Transfer Vector Containing the ORF5 of Porcine Reproductive and Respiratory Syndrome Virus

提取伪狂犬病病毒 ( PRV) Bartha-K6 1株基因组 DNA,用限制性内切酶 Kpn I充分消化 ,回收 5 .9kb片段 ( J片段 ) ,将其克隆于质粒 p UC1 1 9Kpn I位点上 ,获得 p BKJ。用两对针对 PRV TK基因的特异性引物对重组质粒进行 PCR鉴定 ,证明其中含有 PRVTK基因。然后用 Kpn I、Pst I和 Bam HI等限制性内切酶对其进行酶切分析 ,确定了克隆片段的物理图谱。进一步研究证实 TK基因位于其中的 Kpn I- Bam HI或 Kpn I- Pst I片段中。然后亚克隆此 Kpn I- Pst I片段 ,并进行了序列测定 .用 Acc I删去 378bp后 ,并插入CMV启动子控制下的猪生殖 -呼吸道综合征病毒 ( PRRSV) ORF5 ,构建了转移载体质粒p Bd TK- CMV- ORF5。本研究为 PRV活载体的改造和利用奠定了基础。

Pseudorabies virus (PRV) genome DNA was extracted and digested by restriction endonuclease KpnI. The KpnI fragment J of 5.9 kb was shown to contain thymidine kinase (TK) gene by polymerase chain reaction (PCR), and it was cloned to pUC119, resulting a recombinant pBTK5.9. The restriction and PCR analyses confirmed the TK gene was located in its PstI KpnI 2.6 kb fragment. The minor fragment was then subcloned to produce recombinant pBTK2.6. Porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 under the control of CMV promotor was inserted into the pBTK2.6. The resulting transfer plasmid pBdTK CMV ORF5 will be used for devolping TK - and gE - deletd recombinant PRV expressing PRRSV ORF5.