“拯救”NA-1株鹅源副黏病毒辅助质粒的构建及鉴定  

Construction and identification of the helper plasmids for rescuing GPMV strain NA-1

作　　者：宋子运[1] 丁壮[1] 徐明[1] 马爱霞[1] 杜眉[1] 常爽[1] 毕玉海[1] 尹仁福[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国兽医学报》2008年第5期515-517,526,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30571375)

摘　　要：将本实验室保存的NA-1株鹅源副黏病毒(GPMV)经SPF鸡胚增殖,收集尿囊液进行病毒纯化,提取病毒基因组RNA。参考GenBank已收录的ZJ1株GPMV基因组序列,设计了4对特异性引物,利用RT-PCR法分别特异性的扩增出病毒NP、P和L基因片段,并将目的基因片段消化、回收纯化,克隆pCI-neo表达载体,转化大肠杆菌DH5α,小提质粒选取阳性克隆酶切、PCR鉴定并进行序列测定,结果表明NP、P和L基因正确克隆到pCI-neo表达载体中,并分别命名为pCI-NP、pCI-P和pCI-L。将构建好的pCI-NP、pCI-P和pCI-L重组质粒单独转染Vero细胞,在荧光显微镜下观察到特异绿色荧光,表明NP、P和L蛋白得到成功表达。Goose-host paramyxovirus（GPMV）strain NA-1 was separated and conserved by this laboratory. Virus was multiplied by SPF embryonated eggs to collect allantoic fluid in order to obtain the purified virus, and then to extract its RNA genome. Based on the complete genome sequence of goose paramyxovirus strain ZJ1 logged in GenBank and then four pairs of specific primers were designed to amplify NP,P and L gene fragment by the method of RT-PCR. Meanwhile, the target gene fragments were reclaimed and purified to insert into pCI-neo vector to converted into the E. coli DHSα. then the positive clones were identified by enzyme digestion,PCR and sequencing. The recombinant plasmids of pCI-NP,pCI-P and pCI-L were transfected into Vero cells, and the green fluorescence was observed with the method of indirect immunofluorescent test.

关 键 词：鹅源副黏病毒 反向遗传操作技术 辅助质粒 间接免疫荧光 

分 类 号：S852.65[农业科学—基础兽医学]