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Vero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L 4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. Conclusions  Four helper plasmids are constructed genetically and three structural proteins are successfully expressed.