质粒纳米乳剂的制备与阴离子交换色谱测定方法

Preparation of Plasmid Nanoemulsion and Its Character Determination by Anion Exchange Chromatography Method

摘要:

以纳米乳剂为载体包裹草原兔尾鼠卵透明带3 DNA疫苗，制备获得了纳米乳剂DNA疫苗，并对其进行了质量评价。采用界面乳化法制备质粒纳米乳剂，用电子显微镜测定了其粒径及其分布，根据质粒的带电特性，利用强阴离子Q Sepharose^TM XL色谱柱分离纳米乳剂和游离质粒，建立了强阴离子交换柱质粒纳米乳剂包封率的快速测定方法。结果表明：制备的纳米乳剂DNA疫苗的平均粒径为（23±10）nm，包封率为80.5%。选择0.05 mol/L的 Tris-HCl 为平衡液, 流速为0.7mL/min, 紫外检测波长260nm, 柱温30℃, 进样量为2mL的实验条件，质粒的含量和峰面积的线性关系良好（r=0.9983），加样回收率在95%以上，该方法简便快速、灵敏度高，重复性好, 可用于纳米乳剂DNA疫苗包封率的快速测定。

Abstract:

Nanoemulsion-encapsulated lzp3 DNA vaccine（pCDNA3-Aat-COMP-lzp3-C3d3, lzp3） was prepared, and its quality was evaluated. The interfacial emulsification method was employed to make the nanoemulsion-encapsulated plasmid, and its matography was used to separate the nanoemulsion from the plasmid based on the charge characteristics of plasmid. The determination method for encapsulation efficiency of the plasmid nanoemulsion was established by anion exchange chromatography. The results indicated that the average size of the plasmid nanoemulsion is (23±10)nm, and the encapsulation efficiency is 80.5%. The separation and determination conditions of nanoemulsion-encapsulated plasmid was as follows: eluent buffer was 0.05mol/L Tris-HCl solution at a flow rate of 0.7mL/min, while detection wavelength for plasmid was 260nm, the temperature of column was maintained at 30℃, and injection volume was 2mL. On this condition, nanoemulsion and plasmid can be separated on the column of anion exchange chromatography significantly. This method is simple, sensitive and reproducible with respect to good linearity in the range of 0.05～0.80mg/mL (r=0.9983)，which can be applied to determine the entrapment efficiency of nanoemulsion-encapsulated plasmid