重组戊型肝炎病毒P179-海藻酸钠/壳聚糖微球混悬制剂的制备及其免疫效果

目的 制备吸附重组戊型肝炎病毒(hepatitis E virus,HEV)P179抗原的海藻酸钠/壳聚糖微球混悬制剂,并观察其免疫效果。方法 乳化法制备海藻酸钠/壳聚糖微球,采用不同海藻酸钠浓度、混合乳化剂添加量、混合乳化剂亲水亲油平衡值设计正交试验,确定最佳制备工艺参数。将重组HEV P179抗原吸附在最佳工艺制备的微球表面,制备吸附重组HEV P179的海藻酸钠/壳聚糖微球混悬制剂,皮下多点免疫BALB/c小鼠,测定其诱导小鼠产生特异性IgG抗体的能力,与同剂量含弗氏佐剂的重组HEV P179免疫制剂对比。结果 经正交试验确定乳化最佳工艺参数为:海藻酸钠质量分数1%、混合乳化剂体积分数2%,混合乳化剂亲水亲油平衡值4。制备的微球平均粒径为6.73 μm(分布范围3.90~9.10 μm,方差1.78 μm),形态较为均一,透射电镜观察结果与双层球体及内部疏松多孔的微球结构特点相符。用此条件制备抗原质量浓度为500 μg/ml的混悬制剂,微球对抗原的吸附率为90.64%。免疫效果观察试验结果表明,皮下多点免疫试验中微球制剂诱导特异性IgG抗体的能力优于含弗氏佐剂抗原。结论 成功制备了吸附重组HEV P179的海藻酸钠/壳聚糖微球混悬制剂,且诱导产生特异性IgG抗体的能力优于含弗氏佐剂抗原,为其在新型实验动物超敏制剂中的应用奠定了基础。

Preparation and immune effect of recombinanthepatitis E virus p179alginate / chitosan suspension

Objective  To prepare a sodium alginate/chitosan microsphere suspension adsorbing recombinant hepatitis E virus (HEV) P179 antigen and observe its immune effect. Methods  The sodium alginate/chitosan microspheres were prepared by emulsification method. The optimal emulsification preparation process was determined by orthogonal test of different sodium alginate concentrations, mixing emulsifier addition amounts, and mixing emulsifier hydrophile-lipophile balance values (HLBs). The recombinant HEV P179 was adsorbed on the surface of sodium alginate-chitosan microspheres prepared by the optimal process, and the suspension was injected subcutaneously into BALB/c mice. The immunization effect was compared with same dose of recombinant HEV P179 containing Freund's adjuvant for the ability to induce specific IgG antibodies in mice. Results  The optimal process parameters for emulsification were determined to be 1% sodium alginate, 2% mixed emulsifier, and mixed emulsifier HLB of 4. The average particle size of prepared microspheres was 6.73 μm (distribution range 3.90-9.10 μm, variance=1.78 μm), and the shape was relatively uniform. The observation results by radiomicrographs were consistent with the structural characteristics of double-layered spheres and loose and porous microspheres inside. A suspension with 500 μg/ml antigen was prepared, and the adsorption rate of microspheres to antigen was 90.64%. The microsphere preparation showed better ability than antigen with commercial Freund's adjuvant to induce specific IgG antibody in mice. Conclusions  The suspension of alginate-chitosan adsorbing recombinant HEV P179 is successfully prepared, and induces more specific IgG antibodies than same antigen containing Freund's adjuvants. The study lays foundation for the application of this microsphere suspension preparation in a new experimental animal hypersensitivity preparation.