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克隆流行性乙型脑炎(乙脑)病毒野毒(JEV)GSS株前膜蛋白信号序列、前膜蛋白(prM)、包膜蛋白(E)、非结构蛋白-1(NS1)和非结构蛋白NS2a的编码基因,并与非复制型痘苗病毒载体NTV进行同源重组,构建了乙脑病毒非复制型重组痘苗病毒疫苗株NTVA(E/L)JEV。通过PCR和Southernblot检测证明,在非复制型痘苗病毒中有乙脑病毒prM信号序列、prM、E、NS1和NS2a基因的插入:Westernblot检测证明,重组病毒可以在细胞内成功地表达prM、E和NS1蛋白,并可将prM、E和NS1蛋白分泌到细胞培养上清中;免疫荧光检测证明,E和NS1蛋白主要分布在细胞膜上。电镜下可见分泌到细胞外的病毒样颗粒。
Cloning of pre-membrane protein signal sequence, pre-membrane protein (prM), envelope protein (E), nonstructural protein-1 (NS1) Protein NS2a encoding gene and homologous recombination with non-replicating vaccinia virus vector NTV to construct a non-replicating recombinant vaccinia virus vaccine strain NTVA (E / L) JEV. The insertion of prM, E, NS1 and NS2a genes of JEV was confirmed by PCR and Southern blot analysis in non-replicating vaccinia virus: Western blot showed that the recombinant virus could successfully express prM, E and NS1 protein, secreted prM, E and NS1 protein into cell culture supernatant. Immunofluorescence showed that E and NS1 proteins were mainly distributed on the cell membrane. Electron microscopy shows the secretion of extracellular virus-like particles.