The Study Of Structural Activity Of Brain Natriuretic Peptide In Vitro

Brain natriuretic peptide(BNP)is a peptide with biological function in vivo,that consisting of 32 amino acids whose functional domain is a ring-shaped structure consisting of 17 amino acids formed by two cysteine disulfur bonds.Recombinant brain natriuretic peptide(BNP)has been studied as a therapeutic adjunct in acute decompensated congestive heart failure(CHF).BNP binds to natriuretic peptide receptor A(NPR-A)and increases the expression of intracellular cyclic guanosine monophosphate(cGMP).cGMP as a second messenger to activate numerous downstream cascade reactions.One of the degradation pathway for BNP is to be degraded as the substrate of protease in vivo,and the protease include neutral endopeptidease(NEP),dipeptide peptidease 4(DPP4),Trypsinase and MEP1 A.The purpose of this study is to explore the changes in the structure of BNP in vitro and its effecton on activity.The research work includes the following three parts:1)The method of DTT reduction BNP dissulfur bond was established,a stable reduced state BNP sample was prepared,and the structural confirmation of the reduced state BNP was carried out.The active detection of Oxidation and reduction BNP was performed using competitive enzyme-linked immunosorbent Assay(ELISA).The results show that the reductive BNP loses its biological activity after opening the disulfur bond,which proves the important role of BNP disulfur bond structure in its biological activity.2)Using proteinase(DPP4,NEP,MEP1 A and Trypsin)digest BNP in vitro,and confirmed the specificity of the enzyme cutting products.DPP4 and Trypsin obtain a stable specific enzyme dugesting product.This method can be used as the quality standard of BNP peptide mapping and applied to BNP industrialization.The results showed that the activity of DPP4 digested 2-32 BNP was higher than wild-type 1-32 BNP.3)A method of double antibody sandwich ELISA was established to detect the content of DPP4 in plasma.