含LacZ表达盒的鸭瘟病毒TK基因缺失转移载体的构建

Construction of TK Gene Deleted Duck Enteritis Virus Transfer Vector Containing a LacZ Expressing Cassette

根据GenBank已发表的鸭瘟病毒活疫苗C-KCE株基因组序列设计一对引物,扩增出含TK基因完整ORF的2.7 kb的片段,克隆至pMD18T simple载体。利用该片段内的两个酶切位点Xho I和EcoR V,切除TK基因内的244 bp,用T4 DNA聚合酶补平末端;pVAX1-LacZ用Nru I和Nar I双酶切,回收含LacZ表达盒的4.1 kb片段,通过平末端连接将LacZ表达盒插入到TK基因中,从而获得了转移载体pTK-LacZ,为构建重组鸭瘟病毒奠定了基础。

A pair of primer was synthesized according to genome strain C -KCE published on GenBank. It was used to amplify a sequence of duck enteritis virus （DEV） vaccine fragment of 2.7 kb containing the complete TK ORF. The fragment of 2.7 kb was cloned in to pMD18T simple （TaKaRa） to obtain a recombinant plasmid. The recombinant plasmid was digested by Xho I and EcoR V to delete a 244 bp fragment from TK ORF, and then dealt with T4 DNA polymerase for blunting ends. A 4.1 kb LacZ expressing cassette was obtained from pVAX1 - LacZ （Invitrogen） digested by Nru I and Nar I, and cloned in TK gene by joining blunt ends. Then a transfer vector pTK - LacZ was completed, which can lay a foundation for the construction of recombinant DEV