摘要
目的评价毕赤酵母表达的登革病毒1型(DENV-1)病毒样颗粒(VLPs)不同浓度条件下的免疫效果,为登革多价疫苗的研究奠定基础。方法采用毕赤酵母系统构建两种DENV-1 VLPs(即DENV-1 prME-VLPs和DENV-1CprME-VLPs),鉴定、纯化后比较其表达量,选择表达量高的VLPs以不同剂量免疫BALB/c小鼠(试验组分别注射经弗氏佐剂乳化的VLPs 10、25、50μg;对照组分别注射PBS和弗氏佐剂)。通过间接ELISA及中和试验测定免疫小鼠血清特异性抗体效价和中和抗体效价;用VLPs体外刺激免疫小鼠的脾淋巴细胞,MTT法测定其增殖指数;ELISPOT检测IL-4、IFN-γ及TNF-α的抗原特异性淋巴细胞数量。结果以电镜下观察到成功表达的直径约为30nm、形态无明显差异的两种VLPs。两种表达体系中,以DENV-1 prME-VLPs表达量较高。经较高表达量的VLPs免疫小鼠后发现,初次免疫后1周,即可检测到针对DENV-1的抗体,随着时间延长,抗体效价逐渐上升,5周达峰值;其中,VLPs 25μg剂量组与50μg剂量组特异性抗体效价及中和抗体效价均显著高于PBS组、佐剂对照组及VLPs 10μg剂量组,差异有统计学意义(P<0.05)。VLPs 25μg剂量组与50μg剂量组免疫后的小鼠淋巴细胞增殖指数及其分泌IL-4、IFN-γ及TNF-α抗原特异性淋巴细胞数目,均显著高于对照组及VLPs 10μg剂量组,差异有统计学意义(P<0.05)。结论毕赤酵母表达的DENV-1 VLPs免疫剂量为25μg时即可诱导高效价的中和抗体和显著的细胞免疫效应。
Objective To assess the effects of immunity of recombinant dengue virus type-1 （DENV-1） virus-like particles （VLPs） and lay a foundation for developing ployvalent VLPs vaccines. Methods DENV-1 CprME-VLPs （CprME-VLPs） and DENV-1 prME-VLPs （prME-VLPs） were prepared from Pichiapastoris （P.pastoris） expression system, and were purified by sucrose density gradient centrifugation. High-yielded VLPs were selected and used in conjunction with Freund adjuvant （FA） formulations at 10, 25, or 50 μg dose^-1 to vaccine mice. Control animals received phosphate buffered saline （PBS） or FA alone. The mice were tested for both humoral and cellular immune responses by ELISA and virus neutralization test, and by lymphocyte proliferation and detection of the number of IL-4, IFN-T and TNF-α producing cells （IL-4, IFN-γ and TNF-α）. Results Our data demonstrated that CprME-VLPs and prME-VLPs were both spherical particles with a diameter of approximately 30 nm and prME-VLPs were produced in higher yield. Mice immunized with VLPs began to produce antibodies on the day 7 post-prime. The neutralization titers reach 1：40. And the antibody titer and neutralization titers of 25 μg dose^-1 group and 50 μg dose^-1 group were higher than that of the control groups （P〈0.05）, while no difference between the two groups. The proliferation level and detection of the number of IL-4, IFN-γ and TNF-α producing cells from 25 μg dose^-1 group and 50 μg dose^-1 group were higher than that of the control groups （P〈0.05）, while no difference between the two groups. Conclusion Three immunizations with the recombinant DENV-1 VLPs at 25 μg dose^-1 could efficiently induce neutralizing antibodies and cellular immune responses in mice.
出处
《热带医学杂志》
CAS
2014年第7期842-845,863,共5页
Journal of Tropical Medicine
基金
广州市高校科技基金(10A173)
关键词
登革病毒1型病毒样颗粒
中和抗体
抗原特异性淋巴细胞
dengue virus type-1 virus-like particles
neutralizing antibodies
antigen-specific lymphocytes
