马立克病病毒囊膜糖蛋白E基因的克隆及序列分析

CLONING IDENTIFICATION AND SEQUENCE ANALYSIS OF GLYCOPROTEIN E GENE OF MAREK’S DISEASE VIRUS

用聚合酶链式反应 (PCR)技术 ,从中国广西分离的马立克氏病病毒 (MDV)N株和G2 株基因组DNA中 ,扩增出MDV糖蛋白E(gE)抗原基因片段的 1 5 0 0bp编码序列。将PCR扩增产物在KpnⅠ和HindⅢ位点克隆进 pUC1 8质粒载体中 ,以Dig标记的gEPCR产物作为探针做斑点杂交及酶切分析筛选到含MDVgE基因的目的重组质粒 pMNE、pMG2 E。通过酶切位点分析显示 ,两毒株的 gE基因内部酶切位点与MDV GA国际标准强毒株的酶切位点基本一致。对重组 pMNE、pMG2 E质粒进行部分序列测定 ,并用DNAStar软件分析 ,结果表明 :插入序列与发表的GA株gE基因序列具有高度同源性 ,gE基因为高度保守基因。

Glycoprotein E (gE) gene of Marek′s disease virus (MDV) was amplificated from CEF genomic DNA infec\|ted with MDV G 2 and N strain by polymerase chain reaction (PCR). G 2 and N strains were isolated in Guangxi, China. PCR products of the gE gene were labeled with digoxigenin as probes and they were inserted into pUC18 plasmid at the Kpn Ⅰ and Hind Ⅲ sites. Recombinant plasmids were detected by dot\|blotting with the probes of gE PCR products. Restriction endonucleases analysis was used to identify the recombinant plasmid and the recombinant plasmid pMNE, pMG 2E were obtained and the pMNE and pMG 2E gE gene sequences were analyzed by comparing with the published sequence of MDV\|GA strain gE gene, the sequences of N and G 2 strains gE gene were shown that they were almost in accord with the sequence of GA strain gE.