中国血吸虫病防治杂志
中國血吸蟲病防治雜誌
중국혈흡충병방치잡지
CHINESE JOURNAL OF SCHISTOSOMIASIS CONTROL
2010年
1期
5-12
,共8页
王晓婷%朱荫昌%高秋端%郭玲香%李新松
王曉婷%硃蔭昌%高鞦耑%郭玲香%李新鬆
왕효정%주음창%고추단%곽령향%리신송
日本血吸虫病%DNA疫苗%树枝状载体%聚酰胺-胺%PAMAM-Lys
日本血吸蟲病%DNA疫苗%樹枝狀載體%聚酰胺-胺%PAMAM-Lys
일본혈흡충병%DNA역묘%수지상재체%취선알-알%PAMAM-Lys
Schistosomiasis japonica%DNA vaccine%Dendrimer%PAMAM%PAMAM-Lys
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4.0 G PAMAM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/pJW4303SjC23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.
目的 構建併評價一種新型的日本血吸蟲病聚酰胺-胺(PAMAM)型樹枝狀載體DNA疫苗.方法 採用賴氨痠對4.0 G PAMAM進行錶麵脩飾,閤成耑基改性產物PAMAM-Lys.用電泳阻滯實驗確定質粒DNA與PAMAM-Lys複閤的比例,用透射電鏡測試複閤物微觀結構變化,併通過電泳分析複閤物的穩定性.同時採用MTT法對PAMAM-Lys進行體外細胞活性評價.分彆用純化質粒pJW4303、pJW4303-SjC23、樹枝狀載體PAMAM-Lys和複閤物PAMAM-Lys/pJW4303SjC23免疫50隻小鼠,檢測各組小鼠的特異性抗體水平以評價其免疫反應性.結果 瓊脂糖凝膠電泳結果顯示,電荷比在2～4時,PAMAM-Lys與DNA正負電荷完全中和,DNA電泳被完全阻滯,兩者能完全結閤.透射電鏡測試結果錶明,樹枝狀高分子載體與DNA的複閤導緻DNA結構收縮,粒徑減小,分佈均勻.樹枝狀高分子與DNA複閤物具有良好的穩定性.噻唑藍法檢測結果錶明,改性後的樹枝狀高分子載體及其DNA複閤物作用于293T細胞較未改性樹枝狀載體產生的細胞毒性低;經PAMAM-Lys/pJW4303-sj23免疫的小鼠產生的特異性抗體水平顯著高于註射裸DNA疫苗組(P<0.05).結論 氨基痠脩飾的樹枝狀高分子PAMAM-Lys是DNA轉染的優良載體,具有良好的生物相容性,賴氨痠脩飾可以顯著降低PAMAM樹枝狀高分子的細胞毒性,併能增彊DNA疫苗的免疫反應性.
목적 구건병평개일충신형적일본혈흡충병취선알-알(PAMAM)형수지상재체DNA역묘.방법 채용뢰안산대4.0 G PAMAM진행표면수식,합성단기개성산물PAMAM-Lys.용전영조체실험학정질립DNA여PAMAM-Lys복합적비례,용투사전경측시복합물미관결구변화,병통과전영분석복합물적은정성.동시채용MTT법대PAMAM-Lys진행체외세포활성평개.분별용순화질립pJW4303、pJW4303-SjC23、수지상재체PAMAM-Lys화복합물PAMAM-Lys/pJW4303SjC23면역50지소서,검측각조소서적특이성항체수평이평개기면역반응성.결과 경지당응효전영결과현시,전하비재2～4시,PAMAM-Lys여DNA정부전하완전중화,DNA전영피완전조체,량자능완전결합.투사전경측시결과표명,수지상고분자재체여DNA적복합도치DNA결구수축,립경감소,분포균균.수지상고분자여DNA복합물구유량호적은정성.새서람법검측결과표명,개성후적수지상고분자재체급기DNA복합물작용우293T세포교미개성수지상재체산생적세포독성저;경PAMAM-Lys/pJW4303-sj23면역적소서산생적특이성항체수평현저고우주사라DNA역묘조(P<0.05).결론 안기산수식적수지상고분자PAMAM-Lys시DNA전염적우량재체,구유량호적생물상용성,뢰안산수식가이현저강저PAMAM수지상고분자적세포독성,병능증강DNA역묘적면역반응성.
Objective To construct and evaluate a novel PAMAM dendrimer vector-DNA vaccine for schistosomiasis japonica.Methods Lysine was used to modify 4.0G PAMAM.and the modified product PAMAM-Lys was synthesized.Agarose gel electrophoresis was used to confirm the composite ratio of plasmid DNA and dendrimer.Micrestructure 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 agarese 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 counteraeted completely,and the compound was blocked completely by DNA electrophoresis.The obscrvation 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.Thk levels of specific antibodies of the mice immunized with PAMAM-Lys/pJW4303Sj23 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 vacmine which merits further application in schistosomiasis DNA vaccine.
