壳聚糖兽用疫苗稀释液的制备及其应用

Preparation and application of diluent for veterinary chitosan vaccine

目的建立一种壳聚糖稀释液的制备方法,并制备一种生物相容性较好、安全性高、可有效提高疫苗免疫效果的兽用疫苗稀释液,以用于草原牧区大规模动物免疫。方法在酸性条件下溶解壳聚糖,向上清中滴加NaOH使已溶解的可溶壳聚糖析出,制备再生壳聚糖。用不同pH的0.2 mol/L磷酸-柠檬酸缓冲液、醋酸-醋酸钠缓冲液和磷酸盐缓冲液配制1%~5%的壳聚糖以及再生壳聚糖溶液,并在115℃条件下湿热灭菌。通过溶解特性以及外观等条件筛选最佳缓冲体系。利用不同浓度的可溶壳聚糖稀释液(soluble chitosan diluent,SCD)及再生壳聚糖稀释液(regenerated chitosan diluent,RCD)稀释山羊痘活疫苗,通过免疫山羊后的安全性及ELISA抗体水平筛选最佳稀释液配方,并建立最佳稀释液的制备流程。利用制备的稀释液稀释山羊痘活疫苗及猪瘟活疫苗并免疫动物,检测制备的稀释液的安全性及免疫增强作用。结果筛选得到的最佳稀释液配方及其制备过程为:0.2 mol/L pH 4.0醋酸-醋酸钠缓冲液配制3%浓度的壳聚糖溶液,离心去除杂质后,上清滴加NaOH至沉淀完全析出;收集再生壳聚糖,用0.2 mol/L pH 4.6醋酸-醋酸钠缓冲液配制成2%浓度的再生壳聚糖溶液,115℃湿热灭菌30 min;用1 mol/L无菌醋酸钠溶液调pH至6.0并稀释至终浓度0.5%,即为0.5%pH 6.0的RCD。使用该稀释液配制疫苗,仅免疫1次即可达到其他稀释液配制疫苗免疫2次后的抗体水平,免疫增强作用高于铝胶稀释液,同时具备与生理盐水一致的安全性。结论本研究利用再生壳聚糖制备了性状优良、pH接近中性、生物相容性好的兽用疫苗稀释液。基于壳聚糖良好的抗原吸附及缓释特性,稀释液可在疫苗注射剂量不足的情况下达到理想的免疫效果,较为适合大规模动物免疫时使用。

Objective To develop a method for preparation of diluent with good biocompatibility and high safety,which can effectively improve the immune effect of veterinary vaccines and apply to massive animal immunization in pastoral area. Methods Chitosan was dissolved under acidic condition,of which the supernatant was added with sodium hydroxide in droplet to precipitate the soluble chitosan and prepare the regenerated chitosan. The 1% ~ 5% chitosan and regenerated chitosan solutions were prepared with 0. 2 mol/L disodium phosphate-citric acid buffer,phosphate-citrate buffer and acetate-sodium acetate buffer,at various pH values,and sterilized by moist heat at 115 ℃. The optimal buffer system was screened based on the solubility and appearance. Live goat pox vaccine was diluted with various concentration of soluble chitosan diluent(SCD) and regenerated chitosan diluent(RCD) respectively,based on which the formula of diluent was optimized according to the safety and ELISA antibody level of the vaccine in immunized goats,and the preparation process of optimal diluent was developed. Live goat pox vaccine and live swine fever vaccine were diluted with the prepared diluents,with which animals were immunized to evaluate the safety and immunopotentiation of diluent.Results The optimal formula and preparation process of diluent were as follows:chitosan solution at a concentration of3% was prepared with 0. 2 mol/L pH 4. 0 acetic acid-sodium acetate buffer. After the impurities were removed by centrifugation,sodium hydroxide was added to the supernatant until the precipitation was complete. The regenerated chitosan was collected and prepared into a solution at a concentration of 2% with 0. 2 mol/L pH 4. 6 acetic acid-sodium acetate buffer,then sterilized by moist heat at 115 ℃ for 30 min,which was adjusted with 1 mol/L sterile sodium acetate solution to a pH value of 6. 0,and diluted to a final concentration of 0. 5% to obtain the RCD. The antibody level induced by one dose of vaccine prepared with the RCD was equal to that by two doses of vaccine prepared with other diluents as control. The immunopotentiation of RCD was superior to that of aluminum hydroxide gel,while the safety was consistent with that of normal saline. Conclusion The diluent for veterinary vaccines,with good character,nearly neutral pH and good biocompatibility,was prepared by using the recycled chitosan. Due to the good antigen-adsorption and slow-release properties of chitosan,the diluent can achieve an ideal immune effect in case of insufficient vaccine dose,which is more suitable for large-scale animal immunization.