猪链球菌2型MRP和EF双基因共表达重组腺病毒载体的构建  

Construction of Recombinant Adenovirus Vector of Co-expressing MRP and EF Gene from Streptococcus suis Type 2

作　　者：汪涛[1,2] 余辉[1] 梅钧[1] 李兴暖 周小鸥 赵志军[3] 

机构地区：[1]九江学院基础医学院病原生物学教研室,九江332000 [2]江西省系统生物学临床应用重点实验室,九江332000 [3]宁夏医科大学总医院医学实验中心,银川750004

出　　处：《生物技术通报》2013年第8期130-134,共5页Biotechnology Bulletin

基　　金：江西省教育厅科技项目(GJJ12624);九江学院博士科研启动项目(8870909)

摘　　要：旨在构建猪链球菌2型溶菌酶释放蛋白(MRP)和胞外因子(EF)双基因共表达重组腺病毒载体。根据猪链球菌2型MRP和EF的基因序列设计合成2对引物,以猪链球菌2型基因组为模板,扩增MRP基因(第1 801-2 513位)和EF基因(第1 783-2 563位)序列,并克隆到pMD18-T载体。然后将MRP基因、IRES元件和EF基因依次定向克隆至腺病毒穿梭载体pShuttle-CMV中构建重组穿梭质粒pShuttle-CMV-MRP-IRES-EF,再经PmeⅠ酶切,然后转化至含腺病毒骨架质粒pAdEasy-1的BJ5183-AD-1感受态细胞中,经同源重组获得重组腺病毒载体质粒rAdeno-CMV-MRP-IRES-EF。PacⅠ酶切线性化该重组腺病毒载体质粒,再转染AD-293细胞进行病毒包装,最后对细胞包装的病毒液进行PCR鉴定。结果表明,克隆的MRP和EF基因测序正确,酶切重组穿梭质粒和腺病毒载体质粒得到特异酶切产物;线性化的重组腺病毒载体质粒rAdeno-CMV-MRP-IRES-EF转染AD-293细胞6 d后也出现明显的细胞病变(CPE),在培养细胞的上清病毒液中也检测到了MRP和EF基因片段。The purpose of this study was to construct recombinant adenovirus vector of co-expression MRP and EF genes from Streptococcus suistype 2（SS2）.The specific primers were designed by the sequence of MRP and EF genes.The MRP（1 801-2 513）and EF（1 783-2 563）gene fragment were amplified from genomic DNA of SS2 by PCR technique.PCR products were cloned in pMD18-T vector.Then MRP gene,IRES element and EF gene linked into the adenovirus shuttle plasmid（pShuttle-CMV）to construct recombinant shuttle plasmid（pShuttle-CMV-MRP-IRES-EF）.The recombinant shuttle plasmid was lineared with Pme Ⅰ and transformed into BJ5183-AD-1 competent cells to construct homogeneous recombinant adenovirus plasmid（rAdeno-CMV-MRP-IRES-EF）.Then the recombinant adenovirus plasmid was linearized with PacⅠ and transfected into AD-293 cells.Finally,virus liquids of cell pack were identified by PCR.The results showed that MRP and EF genes were verified by gene sequencing.Both the recombinant shuttle plasmid and recombinant adenovirus vector plasmid were verified by enzyme digestion.Cytopathic effect（CPE）was observed after transfection linear rAdeno-CMV-MRP-EF 6 days.MRP and EF gene were also detected in the virus liquid by PCR.

关 键 词：猪链球菌2型 溶菌酶释放蛋白 胞外因子 双基因共表达 重组腺病毒载体 

分 类 号：S852.61[农业科学—基础兽医学] R346[农业科学—兽医学]