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Abstract: Objective To construct the prokaryotic expression system of estrogen receptor α ligand bingding domain (hERα-LBD) and to evaluate the estrogen receptor ligand binding activity of the expressed protein. Methods hERα -LBD was amplicated from the plasmid of hERα -LBD by PCR, the identified PCR product was ligated with pGEM-T-easy vector to generate pGM-T-hERα -LBD. After the confirmation, the hERα -LBD fragments were obtained by enzyme digestion and inserted into pET-28a. The expression vectors were expressed in E.Coli to produce hERα-LBD protein. We mixed the hERα-LBD protein and estradiol and bovine serum albumin conjugated antigens (E2-BSA), then evaluated the binding activity of hERα-LBD by electrophoresis. Results The amplified fragment was about 1.9 kb, which was in agreement with the expected target fragment. Recombinant plasmid of pGM-T-hERα -LBD was confirmed by enzyme digestion and sequencing, then pET-28a(+)-hERα -LBD was constructed successfully. The expressed hERα-LBD protein in E.Coli was observed and the expression amount was 250 mg/L after affinity chromatography purification. hERα-LBD was confirmed to had estrogen binding activity by electrophoresis. Conclusion The prokaryotic expression system of pET-28a(+)-hERα -LBD was successfully constructed, and hERα-LBD had the activity of binding.