I型牛疱疹病毒通用型转移载体的构建

Construction of a Universal Bovine Herpesvirus I Transfer Vector

将 I型牛疱疹病毒 (BHV- 1 ) LA株DNA H ind III A片段中的 Sal I- Sal I亚片段(含 TK基因 )克隆到载体质粒 p BluescriptSK中 ,再用 Bgl II和 Sac I切去 347bp,获得含 TK基因部分缺失的重组质粒 p Sd TK,然后用 H ind III和 Xba I切去其中的多克隆位点 ;将来源于 p CR3- Uni的 CMV启动子、多克隆位点和 BGH poly A信号插入 p Sd TK的Xho I位点上 ,构建了 BHV- 1通用转移载体p Sd TK- CMB,此载体可用来表达牛其它病毒的抗原基因 。

Bovine herpes irus 1 strain LA DNA Hind III A was digested by the restriction endonuclease Sal I,The Sal I subfragment of 2.7kb containing thymidine kinase(TK)gene was cloned into pBluescript SK,resulting in a recombinant pSTK,Then it was digested by Bgl II and Sac I,the 5.31kb fragment was recovered and self ligated,resulting in a recombinant pSdTK,the multiple colning sites were removed by Hind III and Xba I;A fragment containing the immediate early promoter of cytomegalovirus,multiple cloning sites and bovine growth hormone polyadenylation signal derived from pCR3 Uni was amplified and inserted into the Xho I site of pSdTK.The resulting transfer vector pSdTK CMB can be used to expess genes from other bovine viruses(i.e. BPIV3、BRSV、BVDV).