摘要
目的:运用卵黄抗体(IgY)技术制备高效的抗新型冠状病毒(SARS-CoV-2)IgY并检测其免疫学活性。方法:使用SARS-CoV-2灭活疫苗免疫蛋鸡,运用冻融水稀释法、硫酸铵沉淀法和透析法从鸡蛋中提取IgY进行浓缩及纯化并优化IgY提取方法。采用SDS-PAGE对IgY的提取及纯化效果进行表征,BCA法检测纯化后蛋白的浓度、间接ELISA法检测抗体对重组SARS-CoV-2 S1蛋白、N蛋白的效价及其热稳定性。对Balb/c小鼠分别给予15 d常规剂量IgY及30 d大剂量IgY口服及滴鼻处理,采用ELISA法测定小鼠血清对IgY免疫反应性及小鼠体内部分炎症指标IL-1β、IL-6、IFN-γ和TNF-α表达情况,明确IgY安全使用剂量。结果:纯化后IgY SDS-PAGE表征条带正确,IgY提取及纯化效果好。纯化后的IgY效价在免疫完成后第2周达到最高,在200μg/mL时重组SARS-CoV-2 S1蛋白和N蛋白的效价高达1∶256。IgY抗体经25~60℃的温度处理4 h后活性仍基本保持稳定。常规剂量IgY未引起小鼠明显免疫反应。长期大剂量口服及滴鼻IgY时,IgY浓度为20 mg/mL处理组小鼠血清IL-1β、IL-6、IFN-γ及TNF-α分泌显著增加。结论:IgY技术具有快速获得针对不同抗原的高效IgY的潜力,且经其制成的IgY具有良好的热稳定性及生物安全性,但长期使用时IgY剂量应避免超过50 mg/kg,以免加重肾脏处理负担。
Objective:To prepare highly effective anti-SARS-CoV-2 yolk antibody(IgY)using IgY technology and detect its immunological activity.Methods:Laying hens were immunized by the inactivated SARS-CoV-2 vaccine,and IgY was extracted by freeze-thawing water dilution,ammonium sulfate precipitation,and dialysis for concentration and purification,and the IgY extraction method was optimized.The extraction and purification of IgY were characterized by SDS-PAGE,the concentration of the purified proteins was measured by BCA,and the potency of the antibodies against recombinant SARS-CoV-2 S1 and N proteins,as well as their thermal stability,were measured by indirect ELISA.Balb/c mice were given 15 days of a conventional dose of IgY and 30 days of a large dose of IgY through oral and nasal drops,respectively.To confirm the safe dosage range of IgY,ELISA was used to determine the immune reactivity of mice serum to IgY and the expression of IL-1β,IL-6,IFN-γ,and TNF-αin mice.Results:Purificated IgY SDS-PAGE results indicated that the modified extraction and purification of IgY is feasible.The titer of purified IgY reached the highest in the 2nd week after immunization.When the concentration of IgY is 200μg/mL,the titer of IgY against recombinant SARS-CoV-2 S1 protein and N protein reaches 1∶256.The activity of IgY remains stable after treating in water for at 25-60℃4 hours.The conventional dose of IgY can not induce a significant immune response in mice.During the long-term high dose of IgY by oral and nasal administration,20 mg/mL IgY treatment significantly increased the secretion of IL-1β,IL-6,IFN-γ,and TNF-αin the serum of treated mice.Conclusion:IgY technology has the potential to rapidly obtain highly efficient IgY against different antigens,and the IgY produced by it has good thermal stability and biosafety,but the dose of IgY should be avoided to be more than 50 mg/kg for long-term use so as not to aggravate the burden of kidney function.
作者
翟荣云
吴玉娟
许庆安
张玮莹
贲亚琍
樊明文
ZHAI Rongyun;WU Yujuan;XU Qing'an;ZHANG Weiying;BEN Yali;FAN Mingwen(School of Medicine,Jianghan University,Wuhan 430056,Hubei,China;School of Life Science,Jianghan University,Wuhan 430056,Hubei,China;Stomatological Hospital of Jianghan University,Wuhan 430022,Hubei,China)
出处
《武汉大学学报（医学版）》
CAS
2024年第10期1183-1189,共7页
Medical Journal of Wuhan University
基金
中央引导地方科技发展专项资金资助项目(编号:2016ZYYD049)。
