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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DNaseⅠ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 with the proper size and ligate the plasmid to evaluate the binding ability and the protective effect of the chitosan nanoparticles on the plasmid, so as to provide a basis for further study of the immune effect of the recombinant nanovaccine. METHODS: The pIRES-TH-FL-chitosan nanoparticle composite was prepared by ion-crosslinking method. The embedding rate of the nanoparticle to the plasmid was detected by ultraviolet spectrophotometer. The ratio of nanocarrier to plasmid was analyzed by agarose gel electrophoresis Binding ability and the protective effect of the plasmid. The size and shape of the plasmid were observed by gold electron microscopy. The zeta potential and zeta potential were measured by zata potentiometer. Finally, the expression of zeta potential in eukaryotic cells was identified by Western blot. Results: The encapsulation efficiency of chitosan nanoparticles was 99.28%. The results of agarose gel electrophoresis showed that chitosan nanoparticles could protect the plasmid from DNase Ⅰ degradation. The prepared chitosan nanoparticles have a diameter of about 300 nm and a zeta potential of 32.4 mV. Western blot confirmed that the nano-plasmid can be transfected into 293T cells and express the fusion protein of interest. Conclusion: The chitosan nano-particles with proper size and uniform distribution were successfully prepared in this experiment and could be effectively expressed in vitro.