载牛冠状病毒N蛋白壳聚糖微球的免疫效果评价  

Evaluation on the immune efficiency of bovine coronavirus N protein-loaded chitosan microspheres

作　　者：孙庆申[1,2] 李鑫[1] 车小琼[2] 常继涛[1] 杨勇博[2] 于力[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/大动物传染病研究室,黑龙江哈尔滨150001 [2]黑龙江大学生命科学学院/微生物黑龙江省高校重点实验室,黑龙江哈尔滨150080

出　　处：《中国预防兽医学报》2009年第11期882-886,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家"863"计划(2006AA10A203);黑龙江省青年科学基金(QC06C047);哈尔滨市科技创新人才专项基金(2007RFQXN098);黑龙江大学校青年科学基金(QL200643)

摘　　要：为了对壳聚糖分子作为佐剂的缓释效果进行免疫学评价,本研究合成了牛冠状病毒DB2毒株N蛋白基因的3'端第487位～1287位碱基,用大肠杆菌对其进行了高效表达,通过SDS-PAGE电泳分离、电洗脱纯化该牛冠状病毒N蛋白(BCVN)。以戊二醛为交联剂,采用乳化交联的方法制备空白壳聚糖微球。利用吸附法制备载BCVN蛋白的壳聚糖微球,通过肌肉注射途径免疫BALB/c小鼠,用间接ELISA方法检测免疫鼠血清中的抗体水平,由此评价壳聚糖微球对BCVN的缓释作用和佐剂效应。结果表明,壳聚糖微球的形态较好、表面光滑、分散性较好,平均粒径为6.50±1.77μm,电势分布在36mV,吸附BCVN蛋白的壳聚糖微球在体内所产生的免疫效果明显优于唯N蛋白组,说明载BCVN蛋白的壳聚糖微球在体内具有一定的缓释效果,使N蛋白在体内缓慢释放而长时间诱导抗体的产生,该研究结果为以壳聚糖微球作为疫苗佐剂积累了实验数据。A DNA fragment corresponding to the 3′ terminus 487-1 287 bases of the N protein gene of bovine coronavirus DB2 strain were artificially synthesized and expressed in E. coil Blank chitosan microspheres were prepared using glutaraldehyde as crosslinkers followed by emulsification. The expressed BCV N protein was purified by electric elution and loaded on to chitosan microspheres by absorptive method. These microspheres were used to immunize BALB/c mice. The dynamic antibody level in serum was monitored by indirect ELISA to evaluate the slow-release and adjuvanted effect of the BCV N-loaded chitosan microspheres. The results showed that the microspheres had a good shape with smooth surface and an average size of 6.50 ±1.77 μm. The BCV N-loaded chitosan microspheres induced better antibody response than the N protein alone in mice, indicating that the BCV Nqoaded chitosan microspheres had slow-release effect as adjuvant.

关 键 词：壳聚糖 牛冠状病毒核衣壳蛋白 吸附 动物接种 缓释作用 

分 类 号：S852.65[农业科学—基础兽医学]