抗HIV-1 p15(gag)IgY抗体的制备及活性检测  

Preparation of Anti-HIV-1 P 15(gag) IgY and Detection of Its Biological Activity

作　　者：赵文思[1] 邹晓华[2] 沈雪莲[1] 王海龙 柳发勇[1] 刘朝奇[2] 

机构地区：[1]三峡大学医学院,湖北宜昌443002 [2]三峡大学分子生物学研究所,湖北宜昌443002

出　　处：《生物技术》2012年第4期79-83,共5页Biotechnology

基　　金：三峡大学教学研究项目(J2011013)资助

摘　　要：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:The strong specificity and high titer anti-HIV-1p15(gag) IgY was prepared and purified,and its immunological activity was analyzed.Method:Hens were immunized with HIV-1p 15(gag) protein.Water dilution method,ethanol precipitation and salting-out with NaCl were used to extract and purify the IgY.The purification,specificity and titer of the antibody were identified with SDS-PAGE,Western Blot and ELISA,respectively.Result:The HIV-1 p 15(gag)-GST protein was successfully expressed and its molecular weight is 45kDa.After immunized,IgY was obtained.Molecular weight of its heavy chain and light chain is 65kDa and 25kDa,respectively.After the IgY was purified through water dilution method(adjust the pH to 5.1),20%ethanol precipitation and salting-out with 0.028mol/L NaCl.Its purity could be achieved 96% and per milliliter of egg yolk could be gained IgY 9.8mg.The OD450nm value of the IgY was high and stable if it was purified with a final concentration of 18% to 25% ethanol and it has strong specificity and high titer.Conclusion:Purified anti-HIV-1p 15(gag) IgY with high specificity and titer could be obtained after hens were immunized with HIV-1p 15(gag) protein,which provided reliable tools for the future research of HIV-1 and made a solid foundation of IgY using to detect HIV-1.

关 键 词：HIV-1p15蛋白 卵黄抗体 抗体分离纯化 活性检测 

分 类 号：R392.7[医药卫生—免疫学]