Expression of NADP-dependent retinol dehydrogenase/reductase B1 in cervical squamous carcinoma cell line and antibody preparation / 解放军医学杂志

ABSTRACT

Objective The prokaryotic expression vector of NADP(H)-dependent retinol dehydrogenase/reductase(NRDR)B1 was constructed for the detection of NRDRB1 protein,and to prepare its polyclonal antibodies,in order to lay the foundation to study the function of NRDRB1.Methods The coding region of NRDRB1 was constructed to the Gateway-based expression vector(pDEST 14),which was transformed into the Escherichia coli(BL21-AI)for the native protein expression.Overexpression of the recombinant was induced at mid-log growth phase of BL21-AI(A600=0.6)using 0.2% L-arabinose.After supersonication the inclusion bodies of NRDRB1 were purified.New Zealand rabbits were immunized with NRDRB1 as the immunogen,which was recovered from SDS-PAGE gel and subscapsularly injected.The titer of the antiserum was determined by dot blot assay.The antibody was purified by HiTrap Protein G column,and its activity and specificity were assessed by Western blotting and immunohistochemistry.Results The prokaryotic expression vector pDEST 14 with NRDRB1 was constructed.The constructs were sequenced by dideoxynucleotide method.NRDRB1 was overexpressed in strain BL2l-AI.The concentration of recovered NRDRB1 was 0.42mg/ml with a recovery rate of 52.3%.All the immunized rabbits produced high-titer antisera after the second booster.The titer of the antiserum was 1∶2 000 with a detection limit of 6.4ng NRDRB1.The purified antibody had a high specificity.Conclusion The present study provides an effective method of preparing polyclonal antibody against NRDRB1.The purified NRDRB1 native protein and the specific polyclonal antibody of NRDRB1 would be valuable for the study on the biological function of NRDRB1.