猪囊尾蚴cC1蛋白的原核表达及其特异性鸡卵黄抗体的制备与鉴定

Preparation and identification of egg yolk immunoglobulin against cysticercus cellulosae cC1 protein expressed in E.coli

目的制备并鉴定特异性抗猪囊尾蚴鸡卵黄免疫球蛋白(IgY),为猪囊尾蚴病的免疫学诊断提供新的途径。方法原核表达猪囊尾蚴抗原cC1蛋白,纯化后经皮下和肌肉多点注射免疫25周产蛋海兰母鸡,200μg/只,首次免疫28d后加强免疫3次,每次间隔10 d。ELISA法检测IgY抗体效价,Akita法和EGGstractTM IgY Purification System纯化试剂盒分别提取纯化鸡卵黄抗体,考马斯亮蓝法测定抗体含量,Western blot检测抗体特异性。结果 cC1蛋白能有效刺激海兰母鸡产生抗体,首次免疫后第5天左右出现抗体,免疫后第55天为高峰,效价高达1∶105以上,表明制备的鸡卵黄抗体IgY具有较强免疫特异性。结论纯化的猪囊尾蚴cC1蛋白免疫海兰母鸡能产生高效价、特异性的IgY,为进一步研究猪囊尾蚴病的免疫学诊断奠定了实验基础。

Objective To produce and purify egg yolk immunoglobulin（IgY） against cysticercus cellulosae cC1 protein,in order to develop a new way for the immunological diagnosis of cysticercosis.Methods 25-weeks old hens was immunized by subcutaneous injection and intramuscular injection with purified cysticercus cellulosae cC1 protein for 4 times.Each hen was immunized with 200 μg antigen each time.The second injection was performed 28 days later.Subsequent injection were performed at 10 day interval.IgY was extracted from egg by Akita method.EGGstract^TM IgY Purification System was used to purify IgY.ELISA was used to determine the immunoactivities of IgY.The protein concentration of IgY was measured by coomassie brilliant blue method.IgY was analyzed by Western blot.Results IgY could be recognized by cysticercus cellulosae cC1 protein.Specific IgY could be immediately detected by ELISA in the eggs laid by the hens from 5 days after the first immunization.The fastigium of immunoactivity was 55 days after the primary immunization.The antibody tiler overtoped 1 ∶10^5.Yolk IgY had a strong immune specificity.Conclusion The hen can produce high concentration and immunoactivity specific IgY after be immunized with purified cysticercus cellulosae cC1 protein,and it will be useful in the further study of immunological diagnosis of cysticercosis experimental.