【摘要】 目的在组成型的毕赤酵母系统中表达登革2型病毒包膜蛋白E和前膜蛋白prM,并研究病毒样颗粒在酵母细胞中的组装特性。方法以登革2型病毒ZS01/01株mRNA为模板,扩增编码prM/E蛋白的基因。将目的基因克隆至表达载体pGAPZaA的多克隆位点,并转化毕赤酵母GS115。经Zeocin抗生素筛选和PCR鉴定后获得酵母菌重组克隆,并进行表达和鉴定。结果成功克隆登革2型病毒prM/E基因,SDS-PAGE和Western blot检测表明包膜蛋白E在组成型的毕赤酵母系统中得到高水平表达;利用透射电镜在重组酵母胞浆内检测到30-50 nm的登革2型的病毒样颗粒,并发现类似登革病毒感染的双层膜结构;免疫电镜结果证实重组病毒样颗粒可以与抗登革2型病毒血清反应,具有免疫反应性。结论成功的建立了组成型表达登革2型病毒样颗粒的毕赤酵母系统,为登革病毒样颗粒疫苗的制备奠定了基础。更多还原

【Abstract】 The aim of this study was to express the dengue type 2 envelope and pre-membrane proteins in the constitutive Pichia pastoris system,and study the assembly characteristic of virus-like particles in cells.The virus mRNA was taken as the template to amplify the prM/E genes.Then the genes were cloned into the multiple clone sites of the expression vector pGAPZa A,and transformed into Pichia pastoris GS115.By the selection of Zeocin and the identification of PCR,the positive clones was expressed and analyzed.Results demonstrated that dengue virus type 2 prM/E genes were successfully cloned into expression vector;SDS-PAGE and Western blot showed that the envelope protein was high-level expressed in constitutive Pichia pastoris system;the virus-like particles with diameters of 30-50 nm,and the double membrane structures which was similar with the cells infected dengue virus were detected by transmission electron microscopy;results of immune-electron microscopy indicated that the recombinant virus-like particles could be recognized by the anti-dengue type 2 mouse serum,revealed that the particles had immunoreactivity.These results indicate that the constitutive Pichia pastoris system which express dengue type 2 virus-like particles has been established successfully,which will provide the basis for further studies of dengue virus-like particles vaccine.更多还原