抗幽门螺杆菌粘附素HpaA卵黄抗体的制备

目的研制高效价特异性的抗幽门螺杆菌黏附素鸡卵黄抗体免疫球蛋白抗体。方法用纯化重组幽门螺杆菌黏附素(Helicobacter pyloriadhesin A,rHpaA)抗原免疫22周龄罗曼鸡,母鸡免疫应答后,在卵黄中产生抗幽门螺杆菌黏附素抗体;经硫酸铵法初步纯化浓缩后;以间接ELISA鉴定抗体效价及免疫学活性;采用Bradford法测定蛋白含量;并用SDS-PAGE法测定分子量及鉴定抗体纯度。结果母鸡初次免疫第10d即有抗体产生,初次免疫后75d左右抗体效价超过1∶10000;最高可达为1∶12800。卵黄抗体经硫酸铵法纯化后,用SDS-PAGE分析,出现两条蛋白带,纯度达95%以上。其含量为10.56mg/ml蛋黄。结论本研究首次研制并鉴定抗黏附素卵黄抗体,开拓了我国预防幽门螺杆菌感染的新领域,分析了抗体在产蛋期卵黄液中表达的进度,为今后该抗体的持续、高质量诱导奠定基础。有望获得高效价、高产量的抗黏附素的IgY抗体(IgY-HpaA),为制备口服制剂开辟了广阔前景。

Preparation of the anti-yolk sac antibody against Helicobacter pylori adhesin-A

The purified recombinant Helicobacter pylori adhesin-A(HpaA) was used as antigen to immunize Luo-man hens of 22 weeks old together with Freund's adjuvant and the eggs laid by these hens were collected. Then, the yolk sac antibody or yolk immunoglobulin (IgY)against H. pylori adhesin-A was isolated and purified from yolk sac of eggs through water extraction,salt precipitation with ammonium sulfate and dialysis. The antibody titers and its immunological activities were measured by ELISA and its purity was determined by SDS-PAGE. The experimental results showed that the specific Helicobacter pylori adhesin-A-yolk immunoglobulin (HpaA-IgY)could be detected in yolk sacs of eggs laid by hens immunized 10 days after immunization . As demonstrated by ELISA, the titer of IgY in yolk sacs was above 1∶10,000 with a maximal titer of 1∶12 800 and the protein concentration of 10.56 mg/ml. Meanwhile, the purity of the resulting IgY was more than 93% as revealed by SDS-PAGE analysis . It is concluded that the anti-yolk sac antibody against Helicobacter pylori has been firstly and successfully prepared in the present study, thus providing for a useful tool for the studies of H. pylori infections.