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载牛冠状病毒N蛋白壳聚糖微球的免疫效果评价
引用本文:孙庆申,李鑫,车小琼,常继涛,杨勇博,于力.载牛冠状病毒N蛋白壳聚糖微球的免疫效果评价[J].中国预防兽医学报,2009,31(11).
作者姓名:孙庆申  李鑫  车小琼  常继涛  杨勇博  于力
作者单位: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;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
2. 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
3. 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
基金项目:国家"863"计划,黑龙江省青年科学基金,哈尔滨市科技创新人才专项基金,黑龙江大学校青年科学基金 
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(BCV N).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36 mV,吸附BCV N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BCV N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.

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

Evaluation on the immune efficiency of bovine coronavirus N protein-loaded chitosan microspheres
SUN Qing-shen,LI Xin,CHE Xiao-qiong,CHANG Ji-tao,YANG Yong-bo,YU Li.Evaluation on the immune efficiency of bovine coronavirus N protein-loaded chitosan microspheres[J].Chinese Journal of Preventive Veterinary Medicine,2009,31(11).
Authors:SUN Qing-shen  LI Xin  CHE Xiao-qiong  CHANG Ji-tao  YANG Yong-bo  YU Li
Abstract: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 ± 1.77μm. The BCV N-loaded chitosan microspheres induced better antibody response than the N protein alone in mice, indicating that the BCV N-loaded chitosan microspheres had slow-release effect as adjuvant.
Keywords:chitosan  bovine coronavirus N protein  absorption  animal inoculation  slow-release effect
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