登革3型病毒NS1基因重组质粒DNA的构建及其免疫原性观察

目的：构建登革3型病毒NS1基因的真核重组表达质粒，观察其免疫原性，为登革新型疫苗的研究提供依据。方法：从感染鼠脑中提取登革3型病毒RNA，经RT－PCR获得NS1基因片段，然后将其克隆到真核表达载体中。用电穿孔法将重组质粒NDA转入COS7细胞，通过免疫荧光法检测外源基因在真核细胞中的表达，将重组质粒DNA免疫BALB／c小鼠，观察其免疫原性。结果：通过酶切鉴定和序列测定证实了NS1基因片段已经插入真核表达载体，用免疫荧光法证明转染了重组质粒的COS7细胞的胞浆中有特异荧光。重组质粒DNA免疫小鼠后可观察到诱发产生的细胞免疫应答。结论：含有登革3型病毒NS1基因的真核重组表达质粒可以在COS7细胞中进行表达，而且能够诱导小鼠产生细胞免疫应答。

Construction of the recombinant plasmid containing the NS1 gene of dengue 3 virus and observation of its immunogenicity

Objective:To construct a recombinant plasmid DNA containing NS1 gene of dengue 3 virus and observe its immunogenicity in order to lay a foundation for studying a novel dengue vaccine.Methods:Genomic RNA of dengue 3 virus was isolated from murine brain infected with virus. NS1 gene fragment was obtained with RT_PCR, and then cloned into an eukaryotic expressing vector. The recombinant plasmid was transfected into COS7 cells by electroporation and its expression was detected by immuno_fluorescence assay(IFA). Subsequently, the recombinant plasmid DNA was used to immunize BALB/c mice to observe its immunogenicity. Results:With restriction analysis and sequencing, it was confirmed that NS1 fragment of dengue 3 virus was inserted into the vector. The expressed protein could be detected in the cytoplasm of COS7 cells with IFA. The mice immunized with the recombinant plasmid DNA could induce cellular immune response to dengue 3 virus.Conclusion:The recombinant plasmid DNA containing NS1 gene of dengue 3 virus can be expressed in COS7 cells. This kind of recombinant plasmid DNA can induce a specific cellular immune response to dengue 3 virus in mice.