抗日本血吸虫SEA鸡卵黄抗体的制备与鉴定  

Production and Identification of Chicken Egg Yolk Antibodies against Soluble Egg Antigen of Schistosoma japonicum

作　　者：王成祖[1] 莫红梅[1] 程喻力[1] 王磊[1] 蒋自卫[1] 刘文琪[1] 李雍龙[1] 

机构地区：[1]华中科技大学同济医学院病原生物学系,武汉430030

出　　处：《中国寄生虫学与寄生虫病杂志》2007年第3期193-197,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家863高技术研究发展计划(No.2006AA02A251)~~

摘　　要：目的制备特异性抗日本血吸虫可溶性虫卵抗原(SEA)的鸡卵黄免疫球蛋白(IgY)并检测其特异性、敏感性。方法用日本血吸虫SEA经翅膀下静脉和皮下免疫25周龄海兰母鸡4次(首次剂量为60μg/只,加强剂量为30μg/只),每次间隔10d。取免疫前和首次免疫后35d的鸡蛋卵黄,分别用水稀释法提取IgY,BCA法测定蛋白含量,并进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Westernblotting)分析,ELISA检测纯化后IgY特异性和敏感性。分别提取免疫后不同时间的卵黄抗体,观察抗体效价的变化。结果海兰母鸡经SEA首次免疫后35d,每枚蛋经提纯后可得到约61mg抗体,经SDS-PAGE和Westernblotting分析,纯化的IgY有1条主要蛋白带,相对分子质量(Mr)为130000,并可被SEA识别。母鸡初次免疫后第10天,经SDS-PAGE和ELISA分析,鸡蛋卵黄内即有抗体产生,初次免疫后31d效价可达1∶1600。双抗体夹心ELISA结果显示纯化后的IgY有较高的敏感性,可检测到的SEA达2.4ng/ml。结论制备的抗SEA鸡卵黄抗体的特异性和敏感性均较高。Objective To produce and purify egg yolk immunoglobulin against soluble egg antigen （SEA） of Schistosoma japonicum, and evaluate its specificity and sensitivity. Methods 25-week old hen was intravenously and subcutaneously immunized with SEA of Schistosoma japonicum for 4 times. Each hen was first immunized with 60 μg SEA and subsequent injections were performed at 10-day intervals with 30 μg SEA. IgY was extracted from eggs of hen 35 d after the first inoculation by WD （water-dilution） method, eggs from non-immunized hen were used as negative control. The protein concentration of IgY was measured by BCA method, and IgY was analyzed by SDS-PAGE and Western blotting. SEA-based ELISA was used to evaluate the specificity and sensitivity of the IgY. Results 61 mg IgY was extracted from one egg. The results of SDS-PAGE and Western blotting demonstrated that the IgY contained one major protein band with molecular weight of 130 000 and could be recognized by SEA. Specific IgY could be immediately detected by SDS-PAGE and EUSA in the eggs laid by the hens from 10 days after the first immunization. On day 31 after the primary immunization, the antibody titer reached 1：1 600. 2.4 ng/ml SEA was detected by IgY based-sandwich EUSA, which indicated a high sensitivity of the purified IgY. Conclusion Anti-SEA IgY with high specificity and sensitivity has been obtained and purified.

关 键 词：卵黄免疫球蛋白 日本血吸虫 免疫诊断 循环抗原 

分 类 号：R383.24[医药卫生—医学寄生虫学] R392.1[医药卫生—基础医学]