目的:制备适当粒径的壳聚糖纳米粒,并连接质粒,评价该壳聚糖纳米粒对该质粒的结合能力与保护作用,为进一步研究重组纳米疫苗的免疫效果提供基础.方法:采用离子交联法制备成pIRES-TH.FL-壳聚糖纳米粒复合物,用紫外分光光度计检测纳米颗粒对质粒的包埋率;经琼脂糖凝胶电泳分析纳米载体与质粒的结合能力及对该质粒的保护作用;通过喷金电镜观察其大小形态;另用zata电位仪测定粒径和zeta电位:最后用Western blot实验鉴定其在真核细胞中的表达情况.结果:紫外分光光度计检测到该壳聚糖纳米质粒的包埋率为99.28%,琼脂糖凝胶电泳结果显示壳聚糖纳米粒能保护该质粒免受DNase Ⅰ的降解.制得的壳聚糖纳米质粒粒径为300 nm左右.zeta电位为32.4 mV.Western blot证实,该纳米质粒能转染293T细胞并表达目的融合蛋白.结论:本实验成功制备了粒径适当且分布均匀的壳聚糖纳米质粒,并能够在体外实现有效表达.%Objective:To prepare chitosan nanoparticles as gene carrier of multiepitope DNA vaccine,and explore the loading capability and its protection ability to plasmid. Methods:The chitosan nanoparticles were prepared with ionic cross-linking method. The encapsulation rate was determined by UV spectrophotometer. The binding ability and protective effect of plasmid plRES-TH-FL were evaluated by agarose gel electrophoresis analysis. The size,morphology and distribution were observed by transmission electron microscope. The diameter and zeta potential were measured by zetasizer. The pIRES-TH-FL-chitosan nanoparticles were transfected into 293T cells in order to examine their expression. Results:The encapsulation efficiency of pIRES-TH-FI-hitosan nanoparticles was 99.28％ by UV spectrophotometer. Agarose gel electrophoresis showed that chitosan nanoparticles can effectively combined with plasmid plRESTH-FL and protected it from nuclease degradation.The mean diameter of plRES-TH-FL-chitosan nanoparticles was about 300 nm and the mean zeta potential was 32.4mV.Western blot assay result confirmed that the DNA-chitosan nanoparticles can be expressed in 293T cells. Conclusion: pIRES-TH-FL-chitosan nanoparticles of homogeneous diameter were prepared and successfully expressed in vitro.
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