Design, Recombinant Preparation And Activity Evalution Of Natriuretic Peptides

Natriuretic peptide family members structure and biological functions are very similar, They are a class of peptide hormones they have specifically activate receptors with guanylate cyclase activity, They can convert GTP into the second messenger cGMP intracellular, cGMP paly a vital role in cardiovascular and body fluid homeostasis as a part of the effects on body fluid regulation. ANP and BNP analogues at higher doses possess natriuretic, diuretic and cardiac unloading actions but with significant hypotensive properties in the treatment of heart disease. so it increases the risk of the treatment process. CNP possesses more potent antiproliferative and collagen-suppressing properties in cardiac fibroblasts as compared with ANP and BNP. But CNP lacks significant natriuretic and diuretic actions when infused into humans, CNP lacks a C-terminus AA extension, which may explain its lack of natriuretic properties. The natriuretic peptide molecules effect on the function and half-life, becoming a bottleneck in the clinical application.In this thesis new molecular natriuretic peptide, designed and synthesized and their physiological activity are examine, to screen out new molecules with high and comprehensive biological activity. His-tag fusion technology was applied in this paper to produce the VNP. The main conclusions are as follows:Because the member of natriuretic peptides family defects, we design 7 kinds of natriur- etic peptide chimeric, named respectively AnBAc, AnCAc, BnAAc, BnBAc, BnCAc, CnAAc, and CnBAc. Chemical synthesis of these peptides, and detection of these peptides its ability to relax rat vascular, screening out higher physiological activity of new natriuretic peptides than the natural molecules, they are CnAAc, BnAAc, BnBAc and AnCAc, The results laid a foun- dation for the further research and development of the natriuretic peptides.Because of the high cost of chemical synthesis peptides, we explore the genetic recombination technology to prepare peptides. we design the fusion of peptide His6-VNP, and its genetic fragments inserted into Pichia pastoris expression plasmid pPIC9K and pPICZαB, The recombinant plasmid pPIC9K-VNP and pPICZαB-VNP were separately linearized by restriction enzyme SalI and SacI, firstly transformed linear pPIC9K-VNP into Pichia pastoris GS115 by electroporation. Transformants were screened out on G418 plates, then transformed the linearized pPICZαB-VNP into GS115/pPIC9K-VNP, and transformants were screened out on Zeocin plates. Cultured in flask the expression was analyzed by Tricine-SDS-PAGE and Western blot. The results showed that the expression levels of transformants was increased by two antibiotic markers screen. The expression level increased by 75%. Through the nickel ions chelating affinity chromatography purification, obtained the fusion peptide, the biology active of the peptide which prepare by fermention research shows that it possessed inhibiting of the level of macrophage iNOS and TNFαmRNA.