title: [Molecular cloning, prokaryotic expression and double-antibody sandwich ELISA development of 17β-hsd10 in mouse].
abstract: We expressed 17-hydroxysteroid dehydrogenase10 (17β-hsd10) recombinant protein, prepared anti-17β- hsd10 polyclonal antibodies and established sandwich enzyme linked immunosorbent assay (ELISA) test for detection of 17β-hsd10. RT-PCR was used to get the gene of 17β-hsd10 of mouse liver, and a prokaryotic protein expression system pET 15b-17β-hsd10/Escherichia coli BL21 (DE3) which induced with isopropyl-1-thio-β-galactopyranoside (IPTG) for recombinant protein expression was constructed subsequently. The target protein purified using His-Binding-resin column was used to immunize BALB/c mice and rabbits, serum total IgGs from immunized animals were purified by ammonium sulfate precipitation method. We established a Double-antibody Sandwich enzyme linked immunosorbent assay about 17β-hsd10 using the two antibodies we prepared. We got the concentration of 1.5 mg/mL of 17β-hsd10 protein with molecular weight of 29.5 kDa, and polyclonal antibodies from mouse and rabbit with the tite 1.25 x 10(4) and 2.5 x 10(4) respectively. The concentration of 0.1 g/mL of 17β-hsd10 can be detected by the Double-antibody Sandwich ELISA we established, and the assay was sensitive and specific. It can be widely used in clinical and experimental study.
authors.forename: Chuanzhi
authors.initials: C
authors.lastname: Liu
authors.forename: Yingying
authors.initials: Y
authors.lastname: Niu
authors.forename: Yuan'an
authors.initials: Y
authors.lastname: Chen
authors.forename: Cheng
authors.initials: C
authors.lastname: Wu
authors.forename: Yuanhua
authors.initials: Y
authors.lastname: Yu
mesh_terms: D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000235:genetics* / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000276:immunology; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; D015094:3-Hydroxyacyl CoA Dehydrogenases; C458480:Hsd17b10 protein, mouse