Cloning,construction and purification of recombinant human B-type natriuretic peptide

Objective: To clone human B-type natriuretic peptide gene,purify the expressed protein and prepare its polyclonal antibodies.Methods: By using PCR technique,the gene encoding human B-type natriuretic peptide(BNP) was amplified from the cDNA library of human heart and sequenced.Then this gene was inserted into expression vector pGXE4T-2.The construct pGXE4T-2/BNP was expressed in E.coli and the expressed protein was purified by affinity chromatography through GSH-agarose.The purified protein was used to immunize BALB/c mice.Results: The cloned gene was 96 bp in length and its sequence was correct.The construct was expressed in E.coli with a high level of the protein as form soluble,accounting for 19% of the total bacterial proteins.The gene product,characterized by SDS-PAGE and Western blot appeared to be a protein with molecular mass of 29 500.The purity of the protein purified by affinity chromatography reached more than 95%.The titer of anti-sera was 1:32 000 after the fourth immunization.Conclusions: The success of gene clone,expression and purification of human BNP lay the foundation for developing a quick diagnostic kit applying to detecting BNP.