鸡白细胞介素2(IL-2)DNA-壳聚糖纳米粒的制备及体外转染  

Preparation of Chicken Interleukin-2(IL-2) DNA-chitosan Nanoparticles and Its Transfection In vitro

作　　者：张文龙[1,2] 殷喆[1,2] 刘霓红[1] 杨涛[1] 刘胜旺[1] 步志高[1] 王君伟[2] 吴东来[1] 

机构地区：[1]农业部兽医公共卫生重点开放实验室,兽医生物技术国家重点实验室,中国农业科学院哈尔滨兽医研究所,哈尔滨15000 [2]东北农业大学动物医学学院,哈尔滨150030

出　　处：《农业生物技术学报》2009年第4期547-553,共7页Journal of Agricultural Biotechnology

基　　金：国家高科技发展计划(863)项目(No.2006AA10A203)资助

摘　　要：构建了携带鸡白细胞介素2(IL-2)编码基因的重组真核表达质粒,通过复凝聚法制备了含有该重组真核表达质粒的壳聚糖纳米粒子。对制备的IL-2DNA-壳聚糖纳米粒子(IL-2DNA-chitosan nanoparticles)进行了表征。纳米粒子呈球形,粒径分布范围为50～500nm,表面带正电,电势为+17.8mV,DNA质量占纳米粒子总质量的40.2%。DNA酶保护性、稳定性和体外释放试验证明,制备的纳米粒子在微酸性(pH6.0)和微碱性(pH7.4)环境中稳定性较高,可保护携带的DNA分子不被0.6～0.8U/mL DNA酶的降解。用制备的纳米粒子转染Df-1细胞系,间接免疫荧光检测结果证明,该纳米粒子可携带质粒DNA进入细胞,使携带的外源基因获得表达。流式细胞术检测证明,纳米粒子的转染效率为0.2%,与裸DNA转染对照组相比较,包封入壳聚糖纳米粒子中可提高DNA的转染效率。The plasmid carrying chicken interleukin-2 （IL-2） gene was constructed and used to prepare DNA-chitosan nanoparticles by complex coacervation process. The important parameters of the nanoparticles were investigated. The nanoparticles synthesized were globule and the diameter ranged from 50 to 500 nm. The nanoparticles were positively charged with a zeta potential of ＋17.8 mV. DNA counted for 40.2% of the weight of the nanoparticles. Enzyme protection test, stability test and in vitro release test showed that the nanoparticles prepared were stable at pH 6.0 and pH 7.4, and could protect the encapsulated plasmid DNA from nuclease degradation at the nuclease concentration of 0.6-0.8 U/mL. Df-1 cells were transfected by nanoparticles, the result of indirect immuno-fluorescence test demonstrated that the nanoparticles could take the plasmid DNA into cells and the exogenous gene could be expressed by the cells. The transfection efficiency was 0.2% studied by flow cytometry, higher than that of naked DNA transfection control. It demonstrated that encapsulating into nanoparticles can improve the transfection efficiency of DNA.

关 键 词：壳聚糖 纳米粒子 表征 转染 

分 类 号：S188[农业科学—农业基础科学]