重组麻疹病毒沪191株反向遗传系统的建立  

Establishment of reverse genetic system to construct recombinant MV-S191

作　　者：杨志辉 裴捷 郭靖 申硕 YANG Zhihui;PEI Jie;GUO Jing;SHEN Shuo(Research Division 1 of Viral Vaccines,Wuhan Institution of Biological Product Co.Ltd,Hubei 430207,China)

机构地区：[1]武汉生物制品研究所有限责任公司病毒性疫苗研究一室,湖北武汉430207

出　　处：《中国病原生物学杂志》2023年第6期637-642,649,共7页Journal of Pathogen Biology

基　　金：湖北省技术创新专项(重大专项)(No.2017ACA078)。

摘　　要：目的构建麻疹减毒病毒(Measles virus,MV)沪191(MV-S191)株感染性cDNA,以绿色荧光蛋白(eGFP)为靶标,选择外源基因插入区域,建立反向遗传系统重组病毒载体疫苗技术平台。方法提取MV-S191基因组RNA,逆转录获得cDNA,以PCR法分8段扩增麻疹病毒全长基因组cDNA,通过Gibson法连接各片段构建全长基因组cDNA,并将其克隆至pBR322质粒,获得pMV-S191。在pMV-S191的P、M或H、L基因间区域插入eGFP,获得pMV2-eGFP、pMV3-eGFP。同时构建pcDNA-N、P、L3个辅助质粒分别表达麻疹病毒核蛋白(N)、磷酸化蛋白(P)和依赖RNA的RNA聚合酶(L)。将pMV-S191、pMV2-EGFP和pMV3-EGFP分别与辅助质粒通过脂质体lipofectamine 2000共转染BSR-T7/5细胞,5d后取上清及细胞裂解液感染Vero细胞,收获重组病毒。结果通过反向遗传系统成功获得麻疹病毒疫苗株拯救株rMV-S191,感染Vero细胞后,在显微镜下观察到有合胞体形成。通过无缝克隆的方式,将eGFP基因克隆至pMV-S191中,成功获得pMV2-eGFP、pMV3-eGFP,按照相同的方法共转染获得rMV2-eGFP和rMV3-eGFP,将其分别感染Vero细胞,在显微镜下均可观察到形成的麻疹病毒合胞体,且在荧光显微镜下可观察到表达的绿色荧光蛋白。结论成功建立MV-S191的反向遗传系统并确定外源基因的插入位点,为以麻疹病毒为载体新型疫苗的研发奠定了基础。Objective The establishment of the reverse genetic system for MV-S191 is important for basic research and measle virus(MV)based vaccine development.Methods The genomic RNA of MV-S191 was extracted,and the cDNA was obtained by RT-PCR.The GeneArt Gibson Assembly EX Cloning Kits was used to combined the 8 segments amplified from the cDNA.The full-length antisense genome of MV-S191 was amplified and cloned into pBR322 to construct pMVS191.A gene of enhanced Green fluorescent protein(eGFP)was inserted into the region between genes of P and M,H and L of pMV-S191 to construct pMV2-eGFP and pMV3-eGFP.At the same time,three helper plasmids of pcDNA-N,P and L were constructed to express nucleoprotein(N),phosphorylated protein(P)and RNA-dependent RNA polymerase(L)of MV-S191.pMV-S191,pMV2-eGFP or pMV3-eGFP were co-transfected into BSR-T7/5 cells with helper plasmids by lipofectamine 2000.After 5 days post transfection,the harvest(supernatant and cells were)was used to infect(transferred to)Vero cells.Results The measles virus vaccine strain(rMV-S191)was successfully obtained by reverse genetic system.The syncytium formation was observed in vero cells under the microscope.eGFP gene was cloned into pMV-S191 by seamless cloning,and pMV2-eGFP and pMV3-eGFP were successfully obtained.rMV2-eGFP and rMV3-eGFP were obtained by co-transfection according to the same method,and Vero cells were infected respectively.The syncytium could be observed under the microscope,and the expressed green fluorescent protein could be observed under the fluorescence microscope.Conclusion In this study,the reverse genetic system of MV-S191 was successfully established,and the insertion site of foreign gene was determined,which laid the foundation for the research and development of a new vaccine platform based on measles virus vector.

关 键 词：重组麻疹病毒 MV-S191 反向遗传学 T7RNA聚合酶 

分 类 号：R373.1[医药卫生—病原生物学]