呼吸道合胞病毒反向遗传系统中辅助质粒的构建  

Construction of helper plasmids for reverse genetics system of respiratory syncytial virus

作　　者：胡兵[1] 方志正[1] 

机构地区：[1]武汉生物制品研究所有限责任公司病毒疫苗研究室,430060

出　　处：《国际生物制品学杂志》2013年第6期296-299,共4页International Journal of Biologicals

摘　　要：目的 构建可用于表达呼吸道合胞病毒（respiratory syncytial virus,RSV）反向遗传操作中所需的4个功能性结构蛋白的辅助质粒.方法 利用RT-PCR扩增RSV Long株的核蛋白（N）、磷蛋白（P）、大蛋白（L）及转录延长/终止抑制因子M2-1基因.将N、P、M2-1基因PCR产物双酶切后连接表达载体pCI;L基因分两段扩增并分别与pMD 19-T simple载体连接,再先后切下与pCI连接.将构建得到的4个辅助质粒测序并转染Veto细胞,通过间接免疫荧光法检测相应蛋白的表达.结果 扩增得到N、P、M2-1和L4个结构蛋白基因,相应构建的辅助质粒经序列测定,与GenBank公布的RSV Long株序列完全一致;间接免疫荧光法检测表明,N、P、M2-1能在Vero细胞中表达.结论 在分子水平构建了RSV反向遗传学研究中所需的4个辅助质粒,并成功表达出3个结构蛋白.Objective To construct helper plasmids expressing nucleoprotein （N）,phosphoprotein （P）,large protein （L） and transcription elongation/anti-termination factor M2-1 of respiratory syncytial virus （RSV）.Methods N,P,M2-1 genes were amplified by RT-PCR technique.The PCR products were cloned into pCI vectors.L gene was amplified as two fragments which were then cloned into pCI vector,respectively,with help of the intermediate vector pMD 19-T simple.The resultant helper plasmids （pCI-N,pCI-P,pCI-L,pCI-M2-1） were identified by sequencing analysis and transfected into Vero cells.The expressed proteins were detected by indirect immunofluorescence assay （IFA）.Results Four plasmids encoding N,P,L and M2-1 were constructed as demonstrated by sequencing analysis and sequence alignment with RSV Long strain in GenBank.N,P,M2-1 proteins were expressed in Vero cells as confirmed by IFA.Conelusion Four helper plasmids are constructed genetically and three structural proteins are successfully expressed.

关 键 词：呼吸道合胞病毒 质粒 病毒结构蛋白质类 

分 类 号：R373[医药卫生—病原生物学]