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为发展新型疫苗和改造目前使用的麻疹病毒疫苗,以麻疹病毒疫苗株为模板,构建了具有感染性的麻疹病毒cDNA克隆。用RT-PCR分6段扩增出麻疹病毒全长基因,通过酶切、拼接构建麻疹病毒疫苗株CC-47的全长正链cDNA序列,并精确地置于T7启动子控制下与丁型肝炎病毒核酶序列之前。克隆麻疹病毒CC-47株蛋白N、P、L编码区质粒并置于T7启动子控制下,用4个质粒共转染哺乳动物细胞,在表达T7RNA聚合酶的重组痘苗病毒VTF7-3的作用下进行病毒拯救。经免疫荧光、PCR等方法检测证实,获得了具有感染性的麻疹病毒。所拯救的病毒在哺乳动物细胞连续传3代后,仍能检出病毒抗原和核酸。
In order to develop new vaccines and to modify the currently used measles virus vaccine, the infectious measles virus cDNA clone was constructed using the measles virus vaccine strain as a template. The full-length gene of measles virus was amplified by RT-PCR, and the full-length cDNA of measles virus CC-47 was constructed by restriction enzyme digestion and sequencing. The full-length cDNA of measles virus CC-47 was placed under the control of T7 promoter, Hepatitis virus ribozyme sequence before. The plasmids encoding the N, P and L coding regions of the measles virus CC-47 strain were cloned and placed under the control of the T7 promoter. The plasmids were co-transfected into mammalian cells with four plasmids and the effect of recombinant vaccinia virus VTF7-3 expressing T7 RNA polymerase Under the virus rescue. The results of immunofluorescence, PCR and other tests confirmed that the infectious measles virus was obtained. The rescued virus can still detect viral antigens and nucleic acids after three consecutive passages in mammalian cells.