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目的构建并评价一种新型的日本血吸虫病聚酰胺-胺(PAMAM)型树枝状载体DNA疫苗。方法采用赖氨酸对4.0GPAMAM进行表面修饰,合成端基改性产物PAMAM-Lys。用电泳阻滞实验确定质粒DNA与PAMAM-Lys复合的比例,用透射电镜测试复合物微观结构变化,并通过电泳分析复合物的稳定性。同时采用MTT法对PAMAM-Lys进行体外细胞活性评价。分别用纯化质粒pJW4303、pJW4303-SjC23、树枝状载体PAMAM-Lys和复合物PAMAM-Lys/pJW4303-SjC23免疫50只小鼠,检测各组小鼠的特异性抗体水平以评价其免疫反应性。结果琼脂糖凝胶电泳结果显示,电荷比在2～4时,PAMAM-Lys与DNA正负电荷完全中和,DNA电泳被完全阻滞,两者能完全结合。透射电镜测试结果表明,树枝状高分子载体与DNA的复合导致DNA结构收缩,粒径减小,分布均匀。树枝状高分子与DNA复合物具有良好的稳定性。噻唑蓝法检测结果表明,改性后的树枝状高分子载体及其DNA复合物作用于293T细胞较未改性树枝状载体产生的细胞毒性低;经PAMAM-Lys/pJW4303-Sj23免疫的小鼠产生的特异性抗体水平显著高于注射裸DNA疫苗组(P<0.05)。结论氨基酸修饰的树枝状高分子PAMAM-Lys是DNA转染的优良载体,具有良好的生物相容性,赖氨酸修饰可以显著降低PAMAM树枝状高分子的细胞毒性,并能增强DNA疫苗的免疫反应性。
Objective To construct and evaluate a new type of PAMAM dendrimer DNA vaccine against Schistosoma japonicum. Methods 4.0GPAMAM was surface-modified with lysine to synthesize PAMAM-Lys. The ratio of plasmid DNA to PAMAM-Lys was determined by electrophoretic blockade. The microstructure of the composite was tested by transmission electron microscopy. The stability of the complex was analyzed by electrophoresis. MTT assay was used to evaluate the in vitro cell viability of PAMAM-Lys. Fifty mice were respectively immunized with the purified plasmids pJW4303, pJW4303-SjC23, dendrimer PAMAM-Lys and complex PAMAM-Lys / pJW4303-SjC23, and the specific antibody levels of the mice in each group were tested to evaluate their immunoreactivity. Results The results of agarose gel electrophoresis showed that the positive and negative charges of PAMAM-Lys and DNA were completely neutralized when the charge ratio was between 2 and 4, and the DNA electrophoresis was completely blocked. Transmission electron microscopy results showed that the dendrimer complex with DNA resulted in DNA structure shrinkage, particle size reduction, uniform distribution. Dendrimers and DNA complexes have good stability. The results of thiazolyl blue assay showed that the modified dendrimer and its DNA complex had lower cytotoxicity on 293T cells than unmodified dendritic carriers; the mice immunized with PAMAM-Lys / pJW4303-Sj23 The level of specific antibody produced was significantly higher than that of naked DNA vaccine (P <0.05). Conclusion Amino acid-modified dendrimer PAMAM-Lys is an excellent vector for DNA transfection and has good biocompatibility. Lysine modification can significantly reduce the cytotoxicity of PAMAM dendrimer and enhance the immunization of DNA vaccine Reactivity.