麻疹病毒沪191株反向遗传系统的建立

发布时间：2018-11-10 09:05

【摘要】：建立麻疹病毒沪191株(MV-_(S191))反向遗传系统。提取MV-_(S191)的RNA,RT-PCR分7段扩增出麻疹病毒反基因组cDNA,通过酶切、连接,拼接出MV-_(S191)全长反基因组,并分别在其3′端和5′端引入丁型肝炎病毒核酶序列(HdvRZ)和锤头核酸酶序列(HamRZ),克隆到pT7-MCS载体,获得pT7MV_(S191)。在MV-_(S191)的P-M基因间区域插入绿色荧光蛋白(GFP),获得pT7MV_(S191)-ATU-GFP,同时构建表达麻疹病毒核蛋白(N)、磷蛋白(P)和大蛋白(L)的3个辅助质粒。将pT7MV_(S191)、pT7MV_(S191)-ATU-GFP分别与辅助质粒通过脂质体介导共转染BSRT7细胞,热休克3h。转染3d后,取上清,感染Vero-SLAM细胞。经RT-PCR、显微镜检测绿色荧光蛋白表达及合胞体形成,在该系统中成功拯救到两种有感染活性的病毒。成功建立了MV-_(S191)的反向遗传系统,为以麻疹病毒为载体进行新疫苗的研发奠定了基础。
[Abstract]:A reverse genetic system of measles virus strain Shanghai 191 (MV-_ (S191) was established. The RNA,RT-PCR of MV-_ (S191) was extracted and amplified into 7 segments. The anti-genomic cDNA, of measles virus was digested, ligated and spliced into the full-length anti-genome of MV-_ (S191). Hepatitis D ribozyme (HdvRZ) and hammerhead nuclease (HamRZ),) were cloned into pT7-MCS vector at their 3 'and 5' ends, respectively, and pT7MV_ (S191) was obtained. Insertion of green fluorescent protein (GFP), into the P-M region of MV-_ (S191) to obtain pT7MV_ (S191)-ATU-GFP, and construction of nucleoprotein (N), expressing measles virus Three co-plasmids of phosphoprotein (P) and large protein (L). PT7MV_ (S191) and pT7MV_ (S191)-ATU-GFP were co-transfected into BSRT7 cells by liposome and heat shock for 3 h. After 3 days of transfection, the supernatant was taken and infected with Vero-SLAM cells. The expression of green fluorescent protein (GFP) and syncytial formation were detected by RT-PCR, microscope, and two infectious viruses were successfully saved in the system. The reverse genetic system of MV-_ (S191) was successfully established, which laid a foundation for the development of new vaccine based on measles virus.
【作者单位】：成都生物制品研究所有限责任公司;
【分类号】：R373

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