麻疹病毒全长cDNA构建及其感染性的研究

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T7 RNA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.

Construction of Full - length Antigenomic cDNA of Measles Virus Strain CC - 47 and Rescue of Infectious Virus from the cDNA

To establish a reverse genetic system of measles virus from its full-length antigenomic cDNA, six overlapped fragments covering full length cDNA of measles virus CC-47 strain were amplified by RT-PCR.Three plasmids encoding nucleocapsid protein,phosphoprotein and large protein of measles virus were constructed and were under the control of T7 RNA polymerase promoter.The plasmid was constructed containing full-length antigenome cDNA of CC-47 that was under control of T7 promoter and joined with hepatitis delta virus ribozyme sequence.B95a cell was cotransfected with the four plasmids after infection with a helper virus VTF7-3.Rescued CC-47 virus was identified by immunoflurescence, RT-PCR and sequencing.Measles virus was rescued from the system,after three passages of the transfected stock on B95a cell measles virus could still be detected by IFA and RT-PCR.