网络首发:2014-07-20,
纸质出版:2014
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基于DENV-prM/E基因构建DENV-VLP及其免疫学特性[J]. 中山大学学报(医学科学版), 2014,35(4).
Construction and Immunological Properties of Dengue Virus Like Particles Basedon the prM/E Genes[J]. Journal of Sun Yat-sen University (Medical Sciences), 2014, 35(4).
摘 要:【目的】 构建和表达登革病毒4型病毒样颗粒,初步研究其免疫学特性。【方法】 用RT-PCR法扩增DENV4-prM/E基因,构建重组质粒pGAPZ-sprM/E-D4,转化X33酵母菌株,SDS-PAGE、WesternBlot鉴定目的蛋白表达。切片电镜检测病毒样颗粒,蔗糖密度梯度离心纯化。免疫三组Balb/C小鼠,每组15只。采用间接ELISA检测抗原特异性抗体,间接免疫荧光法检测免疫血清与天然病毒粒子的结合,流式细胞术分析小鼠脾淋巴细胞表型,酶联免疫斑点法(ELISPOT)检测脾分泌IFN-γ、TNF-α和IL-10的细胞。【结果】重组质粒在酵母中以病毒样颗粒形式表达,为直径20 ~ 30 nm的球形颗粒,VLP免疫组刺激小鼠产生抗体与灭活病毒组相似,流式及ELISPOT结果提示DENV4-VLP与灭活病毒组相似,用登革病毒抗原刺激后,分泌IFN-γ,TNF-α的细胞数目明显增加,各组分泌IL-10细胞数目的整体水平均不高。【结论】应用毕赤酵母表达系统成功表达获得了DENV4-VLP,具有良好的免疫原性,可诱导Balb/C小鼠产生有效的体液免疫应答,并能诱导Th1型免疫应答而诱导Th2型免疫应答较弱。
Abstract: 【Objective】 To construct and express DENV4-likeparticles and study the immunological properties. 【Methods】 The prM and E genesof DENV4 were amplified by RT-PCR. The recombinant plasmidpGAPZ-sprM/E-D4 was transferredin yeast X33 and expressed. The proteins were identified by SDS-PAGE and Western Blot. The VLP were purified with sucrose densitygradient centrifugation and observed with section electron microscopy. Three groups(15mice in each group) of Balb/c mice were immunized. Antigen specific antibodywas detected by indirect ELISA. Indirect immunofluorescence was used to detect thecombination of serum with natural virus particles. Flow cytometry was used to analysemice spleen lymphocyte phenotype. Enzyme-linked immune spotsmethod (ELISPOT) was used to detect the secreting IFN-γ,TNF-α, and IL-10 spleen cells. 【Results】The recombinant plasmid expressed in the form of virus-likeparticles in the yeast,for 20-30 nm in diameter sphericalparticles. The VLP stimulate antibody production in mice was similar with the inactivatedvirus group. Flow cytometry and ELISPOT results indicated that VLP was similar toinactivated viruses that the number of IFN-γ, TNF-α secreting cells obviously increased while the number of IL-10 secreting cells was not high with dengue virus antigen stimulation.【Conclusion】 The DENV4-VLP has been successfully expressedby Pichia expression system. It showed good immunogenicity, which can induce Balb/Cmice to produce effective humoral immune response and prefer induce Th1 type immuneresponse to Th2 immune response.
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