日本血吸虫可溶性虫卵抗原经两种免疫途径获得的鸡卵黄抗体IgY动态观察

Dynamic Observation of Chicken Egg Yolk Antibodies against Soluble Egg Antigen of Schistosoma japonicum Obtained by Two Immunization Routes

目的对比两种免疫途径产生抗日本血吸虫可溶性虫卵抗原(SEA)的特异性IgY抗体水平动态变化。方法7只新西兰兔感染日本血吸虫尾蚴(1500/只)42d后解剖收集虫卵,制备SEA。将SEA分别经皮下和静脉注射免疫健康海蓝蛋鸡(3只/组),首次和第2次免疫间隔2周,之后每次间隔4周,共免疫5次,50μg/(只·次)。取两组免疫前,以及首次免疫后2～18周生产的鸡蛋,用水稀释法粗提IgY,ELISA检测每2周IgY的动态变化。IgY纯化试剂盒(EGGstract IgY Purifiction System)纯化静脉组首次免疫后8～18周的IgY,紫外吸收法检测抗体浓度,琼脂糖双扩散法和ELISA检测IgY峰值水平的抗体效价,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)分析比较免疫前后抗体特异性。结果静脉组和皮下组分别在首次免疫后8和12周IgY抗体水平达高峰,A492值分别为1.28和0.78,静脉组IgY水平至18周仍维持较高水平,皮下组抗体水平达到峰值后逐渐下降。纯化后IgY浓度约为6.5～9.0mg/ml,琼脂糖双扩散法和ELISA测得静脉注射组峰值水平抗体效价分别为1∶16和1∶51200。经SDS-PAGE和Western blotting分析,纯化后的IgY在相对分子质量(Mr)25000和68000处各有一条清晰条带,且免疫后IgY可与SEA发生特异性反应。结论静脉注射法比皮下注射法能获得更高水平的鸡抗日本血吸虫SEA特异性IgY抗体,且纯化后的IgY抗体具有较好的特异性。

Objective To observe the dynamic changes of the specific chicken egg yolk antibodies （IgY） against soluble egg antigens （SEA） of Schistosoma japonicum by two immunization routes. Methods Seven New Zealand rabbits were infected with S. japonicum cercariae （1 500 per rabbit）. After 42 days the rabbits were sacrificed to collect eggs and prepare SEA. Two groups each with 3 healthy hens were intravenously and subcutaneously immunized with 50 μg SEA, respectively. All hens received five immunizations by the same dose of antigen, with 2-week interval for the first two doses, and 4-week interval for the rest doses. Hen eggs were collected at pre-immunization and every two weeks after the first immunization. Crude IgY was extracted from egg yolk by water dilution method, and were analyzed by SEA-based ELISA, then purified by using EGGstract IgY Purifiction System from the 8th to 18th week after the first immunization. IgY concentration was determined by A260/A280 ratio. The expression of IgY was detected by agarose double diffusion method and SEA-based ELISA. The characteristics of IgY was analyzed by SDS-PAGE and Western blotting. Results The titer of IgY reached a peak at the 8th week in the intravenous group （A492=1.28） and at the 12th week in the subcutaneous group （A492=0.78）, respectively, and maintained at a high level in the intravenous group until the 18th week after the first immunization. The concentration of purified IgY was about 6.5-9.0 mg/ml. Agarose double diffusion method and SEA-based ELISA demonstrated that the peak titer of IgY in the intravenous group was 1 ∶ 16 and 1 ∶ 51 200, respectively. SDS-PAGE demonstrated that IgY contained two major protein bands （Mr 25 000 and 68 000）. IgY purified from immunized egg yolk specifically reacted with SEA. Conclusion The intravenous method is superior than the sub- cutaneous injection method in obtaining a high level of egg yolk antibodies against SEA of Schistosoma japonicum, and the purified IgY shows better specificity.