牛支原体P48蛋白卵黄抗体的制备

Preparation of chicken egg yolk immunoglobulin against P48 protein of Mycoplasma bovis

为了研究牛支原体P48抗原蛋白的免疫学功能及相应卵黄抗体的生物学特性,探究其抗体效价随免疫时间的变化规律,试验采用牛支原体P48蛋白疫苗免疫健康产蛋母鸡,定时采血并收集鸡蛋,用水稀释法结合硫酸铵盐析法制备特异性卵黄抗体,利用间接ELISA法检测血清抗体(IgG)和卵黄抗体(IgY)效价。结果表明,低、中、高剂量组均能诱导蛋鸡产生有效免疫应答,血清抗体效价与卵黄抗体效价的变化趋势基本一致,但卵黄抗体的产生滞后于血清抗体,在首次免疫后8~10周Ig Y效价达到峰值,其中中剂量组的免疫效价最高,最高效价为1:212。经SDS-PAGE检测,制备所得的纯度达86%以上,得率在8.6 mg/m L以上。说明采用牛支原体P48蛋白免疫蛋鸡可制备高效价、高纯度的抗牛支原体P48蛋白卵黄抗体,从而为卵黄抗体在牛支原体的检测与防控方面的研究奠定了基础。

In order to study the immunological function and the biological characteristics of egg yolk immunoglobulin（IgY） against P48 protein of Mycoplasma bovis,laying hens were immunized with P48 protein of M.bovis,and blood and eggs were collected regularly.IgY was isolated and purified by water dilution with ammonium sulfate method,and identified by SDS-PAGE.During immunization,the antibody immune activity and titer of Ig G and IgY were evaluated by enzyme-linked immunosorbent assay（ELISA）.The results showed that the low-,medium-and high-dose groups could induce effectively the production of immune response in laying hens,and the trend of Ig G and IgY titers was uniform,but the production of IgY lagged behind the IgG.The IgY titer reached peak within 8 to 10 weeks after the first immunization,and the highest immune titer was up to 1 ∶ 212 and found in the medium dose group.By SDS-PAGE analysis,more than 8.6 mg of IgY could be gained per milliliter of egg yolk,and the purity was up to 86%.It suggests that IgY with the high titer and purity could be obtained by immunizing chickens with P48 protein of M.bovis,which laid a foundation for application of IgY to detect and control M.bovis infection.