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目的:制备具有高效价强特异性的抗HIV-1 p15(gag)鸡卵黄抗体(IgY),纯化并分析其免疫学活性。方法:用纯化的HIV-1 p15(gag)蛋白抗原免疫蛋鸡,用水稀释法对IgY抗体进行粗提取并结合乙醇沉淀和氯化钠盐析法纯化抗体,再通过SDS-PAGE、Western blot、酶联免疫吸附实验(ELISA)检测抗体的纯度、特异性及效价。结果:表达纯化的HIV-1 p15(gag)-GST融合蛋白分子量为45kDa,用于抗体检测的抗原。用纯化的His-P15蛋白作为免疫原免疫蛋鸡后,获得的卵黄抗体重链、轻链分子量分别为65kDa和25kDa。8倍体积水稀释卵黄,pH值5.1,20%冰乙醇及0.028mol/L盐溶液分离纯化,纯度可达96℅,每毫升卵黄液可得到的卵黄抗体为9.8mg。终浓度为18%～25%的冰乙醇纯化的抗体浓度高且稳定,同时具有较强的特异性和较高的效价。结论:用HIV-1p15(gag)蛋白免疫蛋鸡,可以获得高效价的特异性抗体IgY,为IgY抗体在HIV-1p15蛋白的研究奠定了实验基础。
OBJECTIVE: To prepare anti-HIV-1 p15 (gag) chicken egg yolk antibody (IgY) with high titer and specificity and to purify and analyze its immunological activity. Methods: The chickens were immunized with purified HIV-1 p15 (gag) protein antigen, IgY antibody was purified by water dilution method and purified by ethanol precipitation and sodium chloride salting-out method. SDS- PAGE, Western blot, Enzyme-linked immunosorbent assay (ELISA) was used to detect the purity, specificity and titer of the antibody. Results: The purified recombinant HIV-1 p15 (gag) -GST fusion protein has a molecular weight of 45 kDa and is used as an antigen for antibody detection. Yolk antibody yielded heavy and light chain molecular weight of 65kDa and 25kDa respectively after immunizing laying hens with purified His-P15 protein. Egg yolk was diluted with 8 times volume of water, pH value was 5.1, 20% ice ethanol and 0.028mol / L salt solution was separated and purified with the purity of 96 ℅. The yolk antibody was 9.8mg / ml. The final concentrations of 18% to 25% ice ethanol purified antibody concentration and stability, with strong specificity and high potency. CONCLUSION: Immunization of laying hens with HIV-1p15 (gag) protein can obtain IgY with high titer, which lays the foundation for the research of IgY antibody in HIV-1p15 protein.