摘要目的:研究麻疹病毒(measles virus，MV)沪191株载体中，外源基因插入位置对基因表达及病毒复制的影响。方法:将新型冠状病毒S1蛋白表达序列克隆至MV反基因组不同基因间隔区(N基因前、P和M基因之间、H和L基因之间)，与分别表达T7聚合酶及N、P、L蛋白的辅助质粒共转染293T细胞，转染后细胞裂解上清感染Vero细胞，以获得重组病毒。通过RT-PCR、免疫荧光法、Western blot和ELISA检测S1蛋白，并对重组病毒的增殖特性进行检测。结果:S1克隆至MV N基因前转染未得到重组病毒，克隆至P和M基因之间、H和L基因之间成功获得了重组病毒MV-M-S1、MV-L-S1，并可检测到S1蛋白的表达。MV-M-S1的S1表达量高于MV-L-S1，但病毒滴度低于MV-L-S1。结论:将外源基因插入MV基因组中不同位置，对基因表达及病毒复制将产生不同影响，为后续MV载体的改造提供了经验和参考。

abstractsObjective:To investigate the effects of genomic location of a foreign gene in Shanghai-191 strain measles virus (MV) vector on gene expression and virus replication.Methods:The nucleotide sequence encoding S1 protein of SARS-CoV-2 was inserted at different positions in MV antigenome (the upstream of the N gene, between P and M genes, between H and L genes), and co-transfected into 293T cells with helper plasmids coding T7 RNA polymerase and N, P, and L proteins, respectively. The transfected cells were lysed and the supernatants were used to infected Vero cells to harvest recombinant viruses. S1 proteins expressed by the recombinant viruses were identified by RT-PCR, indirect immunofluorescence assay, Western blot and ELISA. Growth kinetics of the recombinant viruses were analyzed.Results:Recombinant viruses were failed to be rescued when the S1 protein-coding sequence was cloned into the upstream of N gene. Two recombinant viruses, MV-M-S1 and MV-L-S1, were successfully rescued when cloning the S1 protein-coding sequence into the intergenic region between P and M genes, or H and L genes, and could express S1 protein. MV-M-S1 expressed more S1 protein than MV-L-S1, but the titer of MV-M-S1 was lower.Conclusions:Inserting a foreign gene at different positions in the MV genome might have different effects on gene expression and virus replication. This study provided reference for the subsequent construction of MV vector.