日本血吸虫病新型树枝状载体-DNA疫苗的构建及其评价  

Construction and evaluation of a novel dendrimer vector-DNA vaccine for schistosomiasis japonica

作　　者：王晓婷[1] 朱荫昌[1] 高秋端[2] 郭玲香[2] 李新松[2] 

机构地区：[1]江苏省血吸虫病防治研究所、江苏省寄生虫分子生物学重点实验室、江苏省寄生虫病学重点学科,无锡214064 [2]东南大学

出　　处：《中国血吸虫病防治杂志》2010年第1期5-12,共8页Chinese Journal of Schistosomiasis Control

基　　金：卫生部科研基金项目(wkj2006-2-024)

摘　　要：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 novel PAMAM dendrimer vector - DNA vaccine for schistosomiasis japonica. Methods Lysine was used to modify 4.0 G PAMAM, and the modified product PAMAM-Lys was synthesized. Agarose gel electrophoresis was used to confirm the composite ratio of plasmid DNA and dendrimer. Microstructure of the compound was observed by using transmission electronic microscopy, and the stability was analyzed by using electrophoresis. The viability of the cells transfected with dendrimers was evaluated by using a MTT technique in vitro. Fiftyty mice were immunized with purified plasmid pJW4303, pJW4303-Sj23 dendrimer PAMAM-Lys and compound PAMAM-Lys/pJW4303-Sj23, respectively. The specific antibodies of the mice in each group were detected to access the immunoreactivity. Results The agarose gel electrophoresis showed that when the charge ratio of the dendrimer vector and DNA was between 2 and 4, the positive and negative charges could be counteracted completely, and the compound was blocked completely by DNA electrophoresis. The observation results with transmission electronic microscopy showed that the composition of dendrimer vector and DNA caused shrink of DNA structure. Dendrimer-DNA compound had a good stability. MTT showed the modified dendrimer vector and DNA compound system produced a lower cell toxicity on 293T cell than the unmodified ones. The levels of specific antibodies of the mice immunized with PAMAM-Lys/pJW4303- Sj23 were significantly higher than those of the mice immunized with naked DNA vaccine （ P 〈 0.05 ）. Conclusions The lysinemodified PAMAM-Lys is an excellent vector, and has an appropriate biocompatibility. Lysine-modification can reduce the cell toxicity of PAMAM dendrimer significantly. PAMAM-Lys can enhance the immunoreactivity of DNA vaccine which merits further application in schistosomiasis DNA vaccine.

关 键 词：日本血吸虫病 DNA疫苗 树枝状载体 聚酰胺-胺 PAMAM—Lys 

分 类 号：R532.21[医药卫生—内科学]