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目的构建小鼠巨噬细胞炎性蛋白-1α(MIP-1α)的真核表达质粒,观察其作为分子佐剂对单纯疱疹病毒II型(HSV-II)DNA疫苗免疫效果的影响。方法以LPS刺激RAW264.7细胞提取总RNA。采用RT-PCR扩增出MIP-1α基因的全部编码序列,利用克隆载体pUCm-T,将其亚克隆入pcDNA3中,构建出小鼠MIP-1α的真核表达质粒Pm;将其转染COS-7细胞,并用Boyden趋化小室法检测MIP-1α的生物学活性。然后用其与HSV-IIgD的DNA疫苗一起免疫BALB/c小鼠,检测免疫小鼠的特异性抗体、脾T细胞增殖反应及病毒攻击小鼠后对小鼠的保护率,观察MIP-1α对HSV-IIDNA疫苗免疫效果的影响。结果成功构建了小鼠MIP-1α的重组真核表达质粒;免疫BALB/c小鼠发现,其作为分子佐剂可加强HSV-IIgDDNA疫苗的免疫效果。结论小鼠MIP-1α可作为HSV-IIgDDNA疫苗的分子佐剂,为研制新型有效的HSV-IIDNA疫苗提供一定的依据。
Objective To construct an eukaryotic expression plasmid of murine macrophage inflammatory protein-1α (MIP-1α) and investigate the effect of MIP-1α as a molecular adjuvant on the immunogenicity of herpes simplex virus type 2 (HSV-II) DNA vaccine. Methods RAW264.7 cells were stimulated with LPS for total RNA extraction. The full coding sequence of MIP-1α gene was amplified by RT-PCR. The cloned vector pUCm-T was subcloned into pcDNA3 to construct the eukaryotic expression plasmid Pm of mouse MIP-1α. The recombinant plasmid was transfected into COS -7 cells, and the biological activity of MIP-1α was detected by Boyden chemotaxis chamber method. BALB / c mice were immunized with the DNA vaccine of HSV-IIgD, and the specific antibodies of immunized mice, the proliferation of splenic T cells and the protective rate of mice after virus challenge were detected. The effects of MIP-1α pair Effect of HSV-IIDNA Vaccine Immunization. Results The recombinant eukaryotic expression plasmid of mouse MIP-1α was successfully constructed. Immunization of BALB / c mice showed that MIP-1α could enhance the immunogenicity of HSV-IIg DNA vaccine as a molecular adjuvant. Conclusion MIP-1α can be used as a molecular adjuvant for HSV-IIg DNA vaccine and provide a basis for the development of a new and effective HSV-IIDNA vaccine.