高纯度猪血清抗体Fab酶切片段的制备及活性鉴定  

Preparation and activity identification of Fab fragment of high purity porcine serum antibody

作　　者：郎巧利 黄楠 杨希 葛良鹏[1] LANG Qiaoli;HUANG Nan;YANG Xi;GE Liangpeng(Chongqing Academy of Animal Sciences,Chongqing 402460,China)

机构地区：[1]重庆市畜牧科学院,重庆402460

出　　处：《黑龙江畜牧兽医》2022年第14期119-123,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：重庆市农高专项(cstc2020ngzx0001)。

摘　　要：为了从猪血清中分离纯化IgG并制备其Fab片段,试验分别利用Protein G亲和层析法和辛酸-硫酸铵沉淀法(CA-AS法)纯化猪血清中IgG,再通过木瓜蛋白酶水解猪IgG得到Fab片段,并通过Protein A层析柱分离猪Fab和Fc片段,进一步利用凝胶层析纯化猪Fab。通过SEC-HPLC鉴定获得的猪Fab片段的纯度,并通过ELISA和Western-blot鉴定其活性。结果表明:成功从猪血清中纯化得到猪IgG,Protein G亲和层析法较CA-AS法获得的猪IgG纯度更高,纯度>95%,可用于后续猪Fab片段制备试验。木瓜蛋白酶与抗体质量比为1∶10时,水解15 h将猪IgG完全水解。进一步利用Protein A和凝胶层析法纯化猪Fab片段,SEC-HPLC鉴定获得猪Fab片段纯度>99%,ELISA和Western-blot鉴定其活性良好。说明试验成功从猪血清中分离猪IgG,并制备其Fab片段。In order to isolate and purify IgG from porcine serum and prepare for its Fab fragments, Protein G affinity chromatography and octanoic acid ammonium sulfate precipitation(CA-AS) were used to purify IgG from porcine serum. Then, porcine IgG was hydrolyzed by papain to form Fab fragments, and porcine Fab and Fc fragments were separated by Protein A chromatography column, and porcine Fab was further purified by gel chromatography. The purity of porcine Fab fragment was identified by SEC-HPLC, and the activity of porcine Fab fragment was identified by ELISA and Western-blot. The results showed that the porcine IgG was successfully purified from porcine serum, and the purity of Protein G affinity chromatography was higher than that of CA-AS, with a purity of >95%, which could be used for the subsequent preparation of porcine Fab protein. When the enzyme/antibody ratio was 1∶10, the porcine IgG could be completely digested by papain at 15 hours. The porcine Fab fragments were further purified by Protein A and gel chromatography, and the purity of Fab fragment was >99% by SEC-HPLC. ELISA and Western-blot showed that Fab fragment had good activity. It indicated that porcine IgG was successfully isolated from porcine serum and Fab fragment was prepared.

关 键 词：猪 IGG FAB片段 木瓜蛋白酶酶解法 分离纯化 

分 类 号：S852.43[农业科学—基础兽医学]