登革2型病毒ZS01/01株病毒样颗粒免疫原性研究

目的 研究登革2型病毒（Dengue virus type 2,DENV-2）病毒样颗粒（virus-Like particles,VLPs）的免疫原性.方法 利用已构建的DENV-2 ZS01/01株病毒样颗粒的表达质粒转染293T细胞,对分泌型VLPs进行大量培养并通过蔗糖密度梯度离心法对其进行纯化.纯化的VLPs经Western Blot及透射电镜观察等方法鉴定后免疫BALB/c小鼠.利用ELISA及中和试验等方法对体液免疫反应进行检测,ELISPOT法测定细胞免疫水平.结果 登革2型病毒样颗粒表达质粒转染哺乳动物细胞所得上清经蔗糖密度梯度离心后,电镜下可观察到类似于天然登革病毒的大小在45～55nm之间的病毒样颗粒.体液及细胞免疫检测结果显示登革2型VLPs可以刺激小鼠产生较高水平的登革E蛋白特异性抗体及一定水平的中和抗体,免疫小鼠脾淋巴细胞经体外刺激后IFN-γ水平显著升高.结论 登革2型病毒病毒样颗粒免疫BALB/c小鼠后可引起一定水平的细胞免疫及体液免疫反应,该研究结果为四价登革病毒样颗粒疫苗的研制奠定了基础.

Study on the immunogenicity of virus-like particles of dengue virus type 2 ZS01/01 strain

Objective To study the immunogenicity of virus-like particles （VLPs） of dengue virus type 2 （DENV-2） ZS01/01 strain.Methods Secreted VLPs of DENV-2 ZS01/01 strain were purified by ultracentrifugation from 293T cells transfected with plasmid which was established previously in our lab.After identified by Western Blot analysis and transmission electron microscope （TEM）,the immunogenicity of DENV-2 VLPs was evaluated in BALB/c mice.Humoral immune responses were identified by ELISA and CPE-based neutralization analysis while cellular immune responses were detected by ELISPOT.Results The analysis of humoral immune responses revealed that DENV-2 VLPs induced high levels of dengue rEIII-specific IgG antibodies and elicited homotypic neutralizing antibodies against DENV-2 NGC strain.Furthermore,spleen cells from mice vaccinated with DENV-2 VLPs and virions produced comparable levels of IFN-γ.Conclusion Monovalent DENV-2 VLPs produced by mammalian cells were capable to induce dengue-specific humoral and cellular immune responses in mice,and this may open a promising approach to the development of tetravalent dengue VLPs vaccine.