目的 以麻疹病毒（measles virus,MV）沪-191疫苗株MV_S191为载体构建表达风疹病毒（rubella virus，RV）RA27/3疫苗株病毒样颗粒（virus-like particle，VLP）的重组病毒。方法 将RV的开放阅读框2〔包含衣壳（capsid, C）、包膜（envelope，E）1、E2基因〕核酸序列克隆至载体pT7-MV_S191，构建载体pT7-MV_S191-RVS。pT7-MV_S191-RVS与表达MV核蛋白、磷蛋白、大蛋白的3个辅助质粒及表达T7 RNA聚合酶的辅助质粒pCDIBP-T7RNAP共转染BSRT7细胞进行病毒拯救，而后感染Vero细胞完成病毒增殖。采用反转录PCR及基因测序鉴定重组病毒中外源基因的插入；用蛋白质印迹法检测RV C蛋白的表达；通过琼脂糖凝胶过滤层析纯化VLP，蛋白质印迹法分析洗脱峰中VLP的组装；以重组病毒免疫昆明小鼠，采用蛋白质印迹法、ELISA及微量细胞病变抑制法分别检测小鼠血清中抗RV抗体、抗RV抗体滴度及抗MV中和抗体效价。结果 基因测序证明重组病毒中成功插入了表达RV C、E2、E1蛋白的3 189 bp基因片段；蛋白质印迹法可检测到重组病毒中RV C蛋白的表达；分子筛外水洗脱峰中可检测到RV C蛋白。免疫小鼠血清中重组病毒MV_S191-RVS与疫苗株诱导的抗RV抗体滴度相当，滴度达1∶32 000，麻疹中和抗体滴度大于1∶320。结论 利用反向遗传学技术，成功获得表达RV VLP的重组病毒MV_S191-RVS。

Objective To construct a recombinant measles virus （MV） expressing rubella virus (RV) virus-like particle (VLP) using the MV Shanghai-191 vaccine strain (MV_S191) as a vector.Methods The nucleic-acid sequence of RV open reading frame 2, containing capsid (C) and envelope (E) 1/2, was cloned into pT7-MV_S191 vector to construct pT7-MV_S191-RVS. To rescue the recombinant virus, pT7-MV_S191-RVS was co-transfected into BSRT7 cells with 3 auxiliary plasmids expressing MV nucleoprotein, phosphoprotein, and large polymerase, respectively,and pCDIBP-T7RNAP expressing T7 RNA polymerase. Then, Vero cells were infected with the recombinant virus for amplification. Reverse transcription PCR and gene sequencing were used to identify the insertion of exogenous genes in the recombinant virus. Western blot was used to detect the expression of RV C protein. RV VLPs were purified by agarose gel filtration chromatography, and Western blot was used to analyze the assembly of VLPs in the elution peak. Kunming mice were immunized with the recombinant virus, and Western blot, ELISA, and micro-cytopathic effect inhibition assay were used to detect anti-RV antibodies, anti-RV antibody titers, and anti-MV neutralizing antibody titers in mouse serum, respectively.Results Gene sequencing demonstrated that the C-E2-E1 segment of 3 189 bp was inserted into the recombinant virus. Expression of RV C protein was detected by Western blot in the recombinant virus. RV C protein was detected in the void water elution peak. The anti-RV antibody titer induced by recombinant virus MV_S191-RVS in the serum of immunized mice was equivalent to that of RA27/3 vaccine strain, which was 1∶32 000, and measles neutralization antibody titer was ＞1∶320.Conclusion A recombinant virus MV_S191-RVS expressing RV VLP is successfully obtained using reverse genetics technology.