| 摘要: |
| 根据GenBank已发表的鸭瘟病毒(Duck Enteritis Virus, DEV) 活疫苗C-KCE株株基因组序列(Accession No. EU082088),设计一对引物,扩增出含TK基因完整ORF的2.7kbp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 酶切,切除TK基因内的244bp,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4.1Kb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。 |
| 关键词: 鸭瘟病毒 TK基因 LacZ 转移载体 |
| DOI： |
| 投稿时间：2012-04-23修订日期：2012-05-23 |
| 基金项目:科技部科技基础性工作专项“重大动物疫病病原及相关制品标准物质研究（2008FY130100）” |
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| Construction of TK gene deleted DEV transfer vector containing a LacZ expressing cassette |
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| (China Institute of Veterinary Drug Control,Beijing,100081) |
| Abstract: |
| A pair of primer was synthesized according to Duck Enteritis Virus (DEV) vaccine strain C-KCE genome sequence published on GenBank (Accession No. EU082088), and was used to amplify a fragment of 2700 bp containing the complete TK open reading frame. The fragment of 2700 bp 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 in the TK ORF, then dealt with T4 DNA polymerase for blunting ends. A 4.1kb-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. So construction of a transfer vector pTK–LacZ was completed, which was a foundation of a recombinant DEV. |
| Key words: Duck enteritis virus TK LacZ transfer vector |
