title: [Antigen selection, optimized expression and polyclonal antibody preparation of O-GlcNAcase].
abstract: In order to probe the biological function of O-GlcNAc and the pathogenesis of associated diseases, it is essential to prepare a potent and specific O-GlcNAcase (OGA) antibody. Based on protein sequence analysis, we found N terminal 1-350 amino acids of OGA (sOGA) has high antigenicity and hydrophilicity and then constructed it into plasmid pET28a vector. First, we optimized the expression of sOGA in Escherichia coli BL21(DE3) (0.05 mmol/L IPTG, 10 hours) and purified it with the Ni-NTA affinity chromatography and size exclusion chromatography respectively. SDS-PAGE verified the molecular weight (45 kDa) and the purity (>95%) of sOGA and the purified protein was subjected to immunize New Zealand rabbits. Finally, we obtained OGA polyclonal antibody by affinity purifying the antiserum with CNBr-activated Sepharose 4B beads. Western blotting and ELISA assay showed that this antibody could recognize three OGA isoforms with high specificity and the sensitivity was 0.11 ng/mL (the titer was 1:80 000). These results indicated the prepared polyclonal antibody of OGA can be used for the biological function study of OGA.
authors.affiliation: College of Pharmacy and State Key Laboratory of Element-Organic Chemistry, Nankai University, Tianjin 300071, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.forename: Guochao
authors.initials: G
authors.lastname: Li
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.forename: Gaofei
authors.initials: G
authors.lastname: Tian
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Yanling
authors.initials: Y
authors.lastname: Liu
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D017951:Antigen Presentation / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007114:Immunization; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D001619:beta-N-Acetylhexosaminidases / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; C110628:hexosaminidase C; D001619:beta-N-Acetylhexosaminidases