抗非洲猪瘟病毒VP73蛋白鸡卵黄抗体的制备及鉴定  

Preparation and identification of specific chicken egg yolk antibody against VP73 protein of African swine fever virus

作　　者：张彩虹[1] 祝贺[2] 高又文[3] 陶虹[1] 刘建利[1] 宗卉[1] 杨俊兴[1] 曹琛福[1] 曾少灵[1] 阮周曦[1] 吕建强[1] 

机构地区：[1]深圳出入境检验检疫局动植物检验检疫技术中心,广东深圳518010 [2]云南出入境检验检疫局检验检疫技术中心,云南昆明650028 [3]华南农业大学兽医学院,广东广州510642

出　　处：《中国兽医科学》2015年第8期821-825,共5页Chinese Veterinary Science

基　　金：国家高技术研究发展计划(863)项目(2012AA101301)

摘　　要：为研制非洲猪瘟的诊断试剂,将原核表达的非洲猪瘟病毒VP73融合蛋白纯化后,作为抗原免疫蛋鸡,先后用饱和硫酸铵和硫酸钠对卵黄抗体(IgY抗体)进行分离提取,制备抗非洲猪瘟病毒VP73蛋白的特异性IgY抗体。对初步提纯的IgY抗体进行SDS-PAGE和Western-blot鉴定,并用间接ELISA测定IgY抗体的效价。结果显示,提纯后的IgY抗体有1条重链和1条轻链,分子质量分别约为65ku和26ku;IgY抗体可与非洲猪瘟病毒VP73蛋白发生特异性反应,在约71ku处出现特异性杂交条带;间接ELISA测定的IgY抗体效价约为1∶8 192;浓度测定结果显示,每1mL卵黄液可得到9.2mg卵黄抗体。结果表明,用表达的VP73蛋白作为免疫原制备的特异性IgY抗体具有免疫反应原性,可用于研制非洲猪瘟的诊断试剂。In order to develop diagnostic reagents for African swine fever virus（ASFV）,laying hens were immunized with purified ASFV VP73 fusion protein expressed in prokaryotic expression vector and IgY antibody specifically against VP73 protein was purified with saturated ammonium sulfate and sodium sulfate respectively from chicken egg yolk. SDS-PAGE and Western-blot analyses were done with the puri- fied IgY antibody and the titer of the IgY antibody was determined by ELISA. The result showed that the purified IgY antibody had a heavy chain and a light chain,and the relative molecular weight was about 65 ku and 26 ku respectively. The IgY antibody could specifically react with VP73 protein,with a relative mo- lecular weight of 71 ku. Total 9.2 mg IgY antibody could be purified from 1 mL water soluble fraction. The indirect ELISA titer of IgY antibody was about 1 ： 8 192. The above-mentioned results demonstrate that IgY antibody purified from egg yolk of chicken immunized with VP73 protein has biological activity. Combined with specific monoclonal antibody against other proteins of ASFV,IgY antibody can be used to detect ASFV.

关 键 词：非洲猪瘟病毒 VP73蛋白 卵黄抗体 

分 类 号：S852.659.1[农业科学—基础兽医学]