登革3型病毒prM-E基因重组质粒DNA的构建和真核表达

构建登革 3型病毒 prM E基因的真核表达重组质粒 ,并进行体外表达 ,为登革DNA疫苗的研究奠定基础。用RT -PCR法获得 prM -E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DNA转入BHK细胞 ,通过免疫荧光法检测外源基因在真核细胞中的表达。结果 ,通过酶切和序列测定证实了构建的重组质粒DNA含序列正确的 prM- E基因。用免疫荧光法检测到转染了重组质粒DNA的BHK细胞的胞浆中有登革 3型病毒特异蛋白的表达。说明含有登革 3型病毒prM -E基因的真核表达重组质粒可以在BHK细胞中表达 ,该结果为观察该重组质粒的免疫原性奠定了基础。

Construction of the Recombinant Plasmid DNA Containing prM-E Gene of Dengue 3 Virus and Its Eukaryotic Expression

A recombinant plasmid DNA containing prM-E gene of dengue 3 virus was constructed and expressed in vitro, thus lay a foundation for studying dengue DNA vaccines. The prM-E fragment was obtained by RT-PCR and cloned into an eucaryotic eukaryotic expression vector. The recombinant plasmid DNA was transferred into BHK cells by electro-perforation and detected the expression of the exogenous prM-E gene with immuno-fluorescence assay (IFA). The recombinant plasmid DNA was identified with RE digestion analyzing and sequencing. In the cytoplasm of BHK cells infected with the recombinant plasmid DNA, specificity of the expressed product for gene of dengue 3 virus was confirmed by IFA. The recombinant plasmid DNA containing prM-E gene of dengue 3 virus can be expressed in BHK cells lays a foundation for observing the immunogenicity of the recombinant plasmid DNA.