含猪链球菌2型溶菌酶释放蛋白基因片段的重组腺病毒构建与鉴定  

Construction and identification of recombinant adenovirus of muramidase-released protein gene fragment from Streptococcus suis type 2

作　　者：汪涛[1,2] 汤自豪[1] 周小鸥[1] 余辉[1] 周英棠[2] 

机构地区：[1]九江学院基础医学院病原生物学教研室,江西九江332000 [2]九江学院基础医学院江西省系统生物学临床应用重点实验室,江西九江332000

出　　处：《细胞与分子免疫学杂志》2013年第4期350-353,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：江西省教育厅科技项目(GJJ12624);九江学院高层次人才引进科研启动项目(8870909)

摘　　要：目的构建猪链球菌2型(SS2)溶菌酶释放蛋白(MRP)基因片段的重组腺病毒并对其进行鉴定。方法根据MRP的基因序列,设计合成1对引物,以SS2型基因组为模板,扩增MRP基因片段(467-1351)序列。将PCR产物通过pMD18-T载体克隆后,再连接至腺病毒穿梭载体pShuttle-CMV中构建重组穿梭质粒pShuttle-CMV-MRP,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质粒pAdeno-CMV-MRP。PacⅠ酶切线性化该重组腺病毒质粒,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和Western blot法鉴定。结果重组腺病毒质粒pAdeno-CMV-MRP转染AD-293细胞8 d后出现明显的细胞病变,在培养细胞的上清病毒液中也检测到了MRP基因片段及其表达的蛋白。结论成功构建了SS2型MRP基因片段的重组腺病毒(rAdeno-MRP)。Objective To construct and identify the recombinant adenovirus of muramidase-released protein （MRP） gene fragment from Streptococcus suis type 2 （SS2）. Methods The specific primers were designed based on the sequence of MRP gene fragment. The MRP gene fragment （467-1351 bp） was amplified by PCR method with genomic DNA of SS2 as a template. PCR products were cloned in pMD18-T vector. Then MRP gene fragment was linked into the adenovirus shuttle plasmid （pShuttle-CMV） to construct recombinant shuttle plasmid （pShuttle-CMV-MRP）. After Pme I digestion, it was transformed into BJ5183-AD-1 competent cells containing adenoviral backbone plasmid pAdEasy-1 to construct homogeneous recombinant adenovirus plasmid （pAdeno-CMV-MRP）. Then the recombinant adenovirus plasmid was linearized by Pine I and then transfected into AD-293 cells for viral packaging. Finally, the virus liquid was tested by PCR and Westem blotting. Results Cytopathic effect （CPE） was observed at 8 d after transfection of linear pAdeno-CMV-MRP in AD-293 cells. MRP gene fragment and protein expression were also detected in the virus liquid. Conclusion The recombinant adenovirus of MRP gene fragment （rAdeno-MRP） from SS2 was constructed successfully.

关 键 词：猪链球菌2型 溶菌酶释放蛋白(MRP) 重组腺病毒 

分 类 号：R392.33[医药卫生—免疫学] S858.28[医药卫生—基础医学]