In Vitro Study On Influence Of BNP Structure On Bioactivity

Brain natriuretic peptide(BNP)is a polypeptide composed of 32 amino acids which is secreted by cardiac muscle cells.It possesses the property of natriuretic,diuretic,relaxing angio-smooth muscle for decreasing blood pressure,and resisting vasoconstriction of renin-angiotensin-aldosterone system.Recombinant human Brain Natriuretic Peptide(rhBNP)is the in vitro expression outcome of the gene of human Brain Natriuretic Peptide,which is used to treat heart failure.This master thesis is divided into two section.In the first section,Dipeptidyl Peptidase DPP-? present in the blood may specifically cleave BNP,therefore it reduces concentration(activity)of BNP in the blood.The optimum conditions for DPP-? and rhBNP concentration ratio is 1:500,digestion time is 4 h,digestion buffer system fit 20 mmol/l Tris-HCl with PH 7.8,which established the DPP-? endonuclease rhBNP Method.The outcome of rhBNP endonuclease by DPP-? digestion goes through expression and purification steps obtaining a purity of 98 percent of rhBNP in vitro.The HPLC method for detecting DPP-? enzyme product have been established and a purity of 95 percent of 3-32 BNP have been produced.The biological activity of rhBNP was compared with ELISA.The results of three experiments were calculated by statistical t test.P(rhBNP?3-32BNP)=0.0716>0.05.In conclusion,getting rid of the N-terminal amino acid of rhBNP in the structure has no effect on its biological activity.In the second section,we use ELISA and SPR to investigate biological activity of rhBNP production HF-01 and Nesiritide.By using ELISA,The results of three experiments were calculated by statistical t test.P(Nesiritide?HF-01)=0.8496>0.05.ELISA indicates that the bio-generics HF-01 consistent with the original research drug Nesiritide on Biological Activity.We finished SPR methods and searching SPR condition.SPR detection conditions for coupling is 6000 RU.The coupling environment for BNP antibodies and chip surface amino with is that NaAc buffer with 10 mmol/l of pH4.5,Glycine-HCl for chip buffer of pH3.0,analyte concentration greater than 0.39 ?mol/l and smaller than 0.78 ?mol/l.The established SPR method will be used for sample detection analysis.