以壳聚糖纳米微球为载体的Crisp1-DNA避孕疫苗的制备及体外表达

发布时间：2018-03-10 04:33

本文选题：Crisp-　切入点：避孕　出处：《生殖医学杂志》2016年10期 　论文类型：期刊论文

【摘要】：目的构建以壳聚糖纳米微球为载体的Crisp1DNA疫苗,对其物理学、生物学活性进行测定,并评估其在COS-7细胞中的表达情况及细胞毒性。方法采用本实验室前期构建的真核表达质粒pcDNA3.1-Crisp1,以复凝法制备载Crisp1DNA疫苗的壳聚糖纳米粒(CS/DNA NPs),用透射电镜以及凝胶阻滞分析对其相关物理学、生物学活性进行测定,然后将载Crisp1DNA疫苗的壳聚糖纳米粒转染至COS-7细胞,检测其细胞毒性,并用间接免疫荧光法鉴定其在细胞内的表达情况。结果透射电镜结果显示纳米粒形态较均一,平均粒径为189.3nm,Zeta电位约为+0.2mV。凝胶阻滞分析显示壳聚糖微粒可将DNA疫苗完全阻滞于加样孔中并保护DNA质粒不降解。MTT试验显示壳聚糖组细胞存活率显著高于脂质体组(P0.01)。间接免疫荧光实验结果显示CS/DNA NPs能在COS-7细胞中有效表达Crisp 1抗原蛋白。结论由壳聚糖纳米微球介导的Crisp1DNA疫苗,可在真核细胞中有效地表达,且具有较小的细胞毒性,为进一步发展安全有效的DNA避孕疫苗打下了基础。
[Abstract]:Objective to construct a Crisp1DNA vaccine based on chitosan nanospheres and determine its physical and biological activities. Methods the eukaryotic expression plasmid pcDNA3.1-Crisp1, constructed in our laboratory, was used to prepare the chitosan nanoparticles containing Crisp1DNA vaccine. Transmission electron microscopy (TEM) and gel arrest were used to block the expression and cytotoxicity of the recombinant plasmid pcDNA3.1-Crisp1.Methods the expression and cytotoxicity of the recombinant plasmid pcDNA3.1-Crisp1 were evaluated in COS-7 cells. Analysis of its related physics, The biological activity was measured, and then the chitosan nanoparticles loaded with Crisp1DNA vaccine were transfected into COS-7 cells to detect their cytotoxicity. Results the results of transmission electron microscope showed that the morphology of nanoparticles was uniform. The average particle size was 189.3 nm 路DNA potential was about 0.2 MV. Gel block analysis showed that chitosan particles could block the DNA vaccine completely in the sample hole and protect DNA plasmid from degradation. MTT test showed that the cell survival rate of chitosan group was significantly higher than that of liposome group. The results of indirect immunofluorescence assay showed that CS/DNA NPs could effectively express Crisp1 antigen protein in COS-7 cells. It can be expressed effectively in eukaryotic cells and has less cytotoxicity, which lays the foundation for the further development of safe and effective DNA contraceptive vaccine.
【作者单位】：武汉大学人民医院生殖医学中心;
【基金】：国家自然科学基金资助项目(81501306)
【分类号】：R392

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