表达鸡传染性喉气管炎病毒GB基因重组鸡痘病毒转移载体的构建  

Construction of Recombinant Fowlpox Virus Transfer Vector Expressing Glycoprotein B of Infections Laryngotracheitis Virus

作　　者：王晓丽[1] 邵卫星[2] 田夫林 单虎[1] 

机构地区：[1]青岛农业大学,266109 [2]中国动物卫生与流行病学中心,青岛266032 [3]山东省畜牧兽医检验技术中心,济南250022

出　　处：《中国动物检疫》2008年第6期27-30,共4页China Animal Health Inspection

摘　　要：根据文献设计2对引物,PCR扩增FPV复制非必需区中外源基因插入位点两侧翼的片段,分别克隆到pUC19和pSK质粒中,并命名为pFP1和pFP2。根据文献设计引物,PCR扩增鸡传染性喉气管炎病毒(ILTV)的GB基因的编码区。将GB基因克隆到质粒pFP1中获得重组质粒pFP1-GB;用酶切质粒pEGFP,得到GFP片段并克隆到质粒pFP2中,得到重组质粒pFP2-GFP。双酶切pFP1-GB得到FP1-GB片段,并克隆到质粒pFP2-GFP中,得到重组质粒pFP1GB-FP2GPF。酶切质粒pE/L-7.5,获得背向连接的2个鸡痘病毒启动子片段——PE/L-7.5,并将该片段克隆到重组质粒pFP1GB-FP2GPF获得重组质粒pGB-GFP,该质粒即为鸡痘病毒的转移质粒。在质粒pGB-GFP中,2个背向连接的启动子被克隆到的GB和GFP基因之间,分别启动GB和GFP基因的表达。经酶切和测序鉴定pGB-GFP,启动子PE/L和P7.5已分别正确地插入到GB和GFP基因之间,分别启动外源表达基因GB和筛选标记基因GFP的表达,从而证明已获得表达鸡传染性喉气管炎病毒GB基因重组鸡痘病毒转移载体pGB-GFP。该载体为下一步获得表达鸡传染性喉气管炎病毒GB基因的重组鸡痘病毒奠定基础。Two the primers two pairs of primers were designed according to the fowlpox gene fragments in the nonessential region of FPV were amplified by sequence in the genbank, and with PCR and then cloned into pUC19 and pSK vectors, named pFP1 and pFP2. At the same time a 2.7Kb DNA fragment encoding glycoprotein B （GB） of infections laryngotraccheitis virus was obtained by PCR with another primers, then was cloned into pFP1 to obtain pFP1-GB. The plasmid pEGFP was digested by restriction enzymes, GFP fragment was recovered and then was cloned into pFP2, named pFP2-GFP. FP1-GB fragment, obtained by digesting pFP1-GB with two different restriction enzymes, was cloned into pFP2-GFP, named pFP1GB-FP2GFP. The pE/L-7.5 promoter fragments containing two reversed connection FPV promoters, were cloned into pFP1GB-FP2GFP, and the recombinant plasmid pGB-GFP was constructed. It was tested that the promoters, reversed connection, were just inserted in the middle of GB and GFP by restriction enzyme digesting test and sequence analysis, so it was identified that the transfer vector pGB-GFP expressing GB of ILTV was successfully constructed. In the vector ,the exogenous expression gene and the selective marker gene were promoted by the pE/L and p7.5 correspondingly.

关 键 词：鸡传染性喉气管炎病毒 糖蛋白GB基因 重组鸡痘病毒 转移载体 

分 类 号：S852.65[农业科学—基础兽医学] Q782[农业科学—兽医学]