Glycosylation of recombinant human thyroid peroxidase ectodomain of insect cell origin has little effect on recognition by serum thyroid peroxidase antibody  

作　　者：LILT Ming-ming LI Qing ZHAO Lan-lan GAO Ying HUANG You-yuan LU Gui-zhi GAO Yan-ming GUO Xiao-hui SHI Bing-yin 

机构地区：[1]Peking Univ, Hosp 1, Dept Endocrinol, Beijing 100034, Peoples R China [2]Chinese Acad Sci, Inst Microbiol, CAS Key Lab Pathogen Microbiol & Immunol, Beijing 100101, Peoples R China [3]Xi An Jiao Tong Univ, Sch Med, Affiliated Hosp 1, Dept Endocrinol, Xian 710061, Shaanxi, Peoples R China

出　　处：《Chinese Medical Journal》2013年第15期2907-2911,共5页中华医学杂志（英文版）

摘　　要：Background Thyroid peroxidase （TPO） is an important autoantigen in Hashimoto＇s thyroiditis （HT）, and almost all epitopes are located in TPO ectodomain. The glycosylation of TPO might contribute to breaking self-tolerance, therefore, pudfied glycosylated recombinant TPO ectodomain is prerequisite of elucidating its role in the pathogenesis of HT. The aim of our study was to investigate whether the glycosylation has influence on the antigenic determinants of recombinant TPO. Methods Bac-to-Bac baculovirus expression system was used to generate recombinant human TPO ectodomain. The antigenicity was analyzed by antigen specific enzyme-linked immunosorbant assays （ELISAs）. The glycosylation of recombinant human TPO ectodomain of High Five insect cell origin was detected by lectin-ELISAs. Results TPO ectodomain was recovered from the culture media as a soluble protein, and it was fused with a hexahistidine tag which allowed purification by nickel-affinity chromatography. The recombinant TPO ectodomain could be recognized by all the 54 HT patients and three TPO monoclonal antibodies. Fucose, sialic acid and galactose were all detected on the recombinant TPO ectodomain. Sera TPOAb binding decreased slightly after non-specific deglycosylation of TPO by periodic acid. Conclusions High Five insect cells derived recombinant human TPO ectodomain had N-glycosylation sites, which might have little effect on recognition by serum TPOAb.Background Thyroid peroxidase （TPO） is an important autoantigen in Hashimoto＇s thyroiditis （HT）, and almost all epitopes are located in TPO ectodomain. The glycosylation of TPO might contribute to breaking self-tolerance, therefore, pudfied glycosylated recombinant TPO ectodomain is prerequisite of elucidating its role in the pathogenesis of HT. The aim of our study was to investigate whether the glycosylation has influence on the antigenic determinants of recombinant TPO. Methods Bac-to-Bac baculovirus expression system was used to generate recombinant human TPO ectodomain. The antigenicity was analyzed by antigen specific enzyme-linked immunosorbant assays （ELISAs）. The glycosylation of recombinant human TPO ectodomain of High Five insect cell origin was detected by lectin-ELISAs. Results TPO ectodomain was recovered from the culture media as a soluble protein, and it was fused with a hexahistidine tag which allowed purification by nickel-affinity chromatography. The recombinant TPO ectodomain could be recognized by all the 54 HT patients and three TPO monoclonal antibodies. Fucose, sialic acid and galactose were all detected on the recombinant TPO ectodomain. Sera TPOAb binding decreased slightly after non-specific deglycosylation of TPO by periodic acid. Conclusions High Five insect cells derived recombinant human TPO ectodomain had N-glycosylation sites, which might have little effect on recognition by serum TPOAb.

关 键 词：thyroid peroxidase baculovirus expression vector system sialic acid GALACTOSE FUCOSE 

分 类 号：Q572[生物学—生物化学] TQ467.32[化学工程—制药化工]