抗单纯疱疹病毒1型的IgY制备及其生物学活性检测  

Preparation of IgY against herpes simplex virus-1 and detection of its biological activity

作　　者：苏海涛[1] 翟玥[2] 宋秀玲[2] 徐坤[2,3] SU Haitao;ZHAI Yue;SONG Xiuling;XU Kun(Institute of Parasitic Disease Prevention and Control,Center for Disease Control and Prevention of Jilin Province,Changchun 130062,China;Department of Hygienic Inspection,School of Public Health,Jilin University,Changchun 130021,China;Department of Food and Nutrition Hygiene,School of Medical Sciences,Hunan Normal University,Changsha 410013,China)

机构地区：[1]吉林省疾病预防控制中心寄生虫病预防控制所,吉林长春130062 [2]吉林大学公共卫生学院卫生检验教研室,吉林长春130021 [3]湖南师范大学医学院食品与营养卫生教研室,湖南长沙410013

出　　处：《吉林大学学报（医学版）》2024年第2期303-309,共7页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科技创新基地(平台)建设项目(YDZJ202302CXJD054)。

摘　　要：目的:制备抗单纯疱疹病毒1型(HSV-1)的卵黄抗体(IgY),探讨该抗体的生物学活性,阐明其抗HSV-1的能力。方法:制备HSV-1灭活疫苗,采用鸡胸多点注射法免疫高产蛋鸡,采用聚乙二醇-6000(PEG-6000)法提纯IgY,采用间接ELISA法、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术和蛋白浓度定量试剂盒测定IgY效价、纯度及蛋白水平,采用间接ELISA法检测IgY中和病毒的能力,测定病毒阻断率,以Vero细胞为病毒感染靶细胞,测定不同抗体浓度(0.01560、0.03125、0.06250、0.12500、0.50000、和1.00000 g·L^(-1))作用后细胞病变效应(CPE),根据CPE程度测定IgY体外抗HSV-1能力。结果:制备的IgY效价随免疫时间逐渐升高,达到1/1024000后保持稳定;IgY轻链重链条带清晰;IgY平均蛋白水平为11.544 g·L^(-1);IgY对HSV-1的阻断率可高达77.90%;HSV-1致CPE程度随抗体浓度升高而逐渐降低,当IgY浓度达到0.50000g·L^(-1)及以上时,25%以下细胞(甚至无细胞)发生病变。结论:成功制备出抗HSV-1的IgY,该抗体对HSV-1具有明显的中和阻断能力和体外抑制活性,可用于药物及诊断方法的开发。Objective:To prepare the anti-herpes simplex virus-1(HSV-1)egg yolk antibody(IgY)and discuss its biological activity,and to clarify its anti-HSV-1 capability.Methods:The inactivated HSV-1 vaccine was prepared,the high egg-producing hens were immunized by multi-site injection method in the chicken breast,and IgY was purified by polyethylene glycol-6000(PEG-6000)method;the titer,purity,and protein levels of IgY were detected by indirect ELISA method,sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)technology,and protein concentration quantification kits;the ability of IgY to neutralize the virus was detected by indirect ELISA method;the virus blocking rate was detected.The Vero cells were used as the target cells for viral infection.The cytopathic effect(CPE)at different antibody concentrations(0.01560,0.03125,0.06250,0.12500,0.25000,0.50000,and 1.00000 g·L^(-1))was detected;the in vitro anti-HSV-1 capacity of IgY was detected according to the degrees of CPE.Results:The titer of the prepared IgY was gradually increased with the extension of immunization time.The titer of the IgY stabilized at 1/1024000;the clear bands for the IgY light and heavy chains were observed;the average protein level of IgY was 11.544 g·L^(-1);the blocking rate of IgY against HSV-1 could reach up to 77.90%;the degree of HSV-1 induced CPE was decreased with the increasing of the antibody concentrations,and when the concentration of IgY reached 0.50000 g·L^(-1)or higher,less than 25%of the cells(even no cells)exhibited cytopathic changes.Conclusion:Anti-HSV-1 IgY is successfully prepared,and this antibody has significant neutralizing and blocking capabilities and in vitro inhibitory activity against HSV-1,which can be utilized for the development of drugs and diagnostic methods.

关 键 词：单纯疱疹病毒1型 卵黄抗体 中和病毒 体外抗病毒能力 

分 类 号：S852.43[农业科学—基础兽医学]