Molecular cloning, prokaryotic expression and double-antibody sandwich ELISA development of 17β-hsd10 in mouse.
- Author:
Chuanzhi LIU
;
Yingying NIU
;
Yuan'an CHEN
;
Cheng WU
;
Yuanhua YU
- Publication Type:Journal Article
- MeSH:
3-Hydroxyacyl CoA Dehydrogenases;
genetics;
immunology;
Animals;
Antibodies;
immunology;
Cloning, Molecular;
Enzyme-Linked Immunosorbent Assay;
methods;
Escherichia coli;
Immunization;
Mice;
Mice, Inbred BALB C;
Rabbits;
Recombinant Proteins;
genetics;
immunology
- From:
Chinese Journal of Biotechnology
2014;30(11):1774-1780
- CountryChina
- Language:Chinese
-
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.
