Recombinant Preparation And Preliminary Activity Evaluation Of Vascular Natriuretic Peptide

Natriuretic peptide family is a peptide family recently discovered which has a ring of 17-amino acid domain with a disulfide bond formed by two cysteines. Natriuretic peptide family plays an important role in the maintenance of water and salt balance, blood pressure stability, cardiovascular, renal and other organ function in vivo. Vascular natriuretic peptide (VNP), which was artificially designed, was composed of the function domain of C-type natriuretic peptide(CNP) and A-type natriuretic peptide(ANP) with the expectation to retain both advantages of CNP and ANP.The VNP gene was firstly amplified by PCR and then was purified. Plasmid pGEX-4T-1 and the PCR product were both digested by EcoR I and Not I. After that, they were linked together and VNP gene was successfully cloned into the expression vector pGEX-4T-1. Till then the recombinant plasmid pGEX-4T-1-VNP was finally constructed. We made use of colony PCR method to identify the recombinant plasmid. Besides that, the sequencing result showed that the cloned gene sequences were exactly the same with the expected VNP gene sequences.The recombinant plasmid pGEX-4T-1-VNP was transformed into E.coli BL21 (DE3). It was found that by using IPTG to induce the expression of fusion protein GST-VNP, the yield was higher when under lower temperature. Furthermore, GST-VNP was soluble intracellular expression. Through GST affinity chromatography we easily obtained fusion protein GST-VNP with purity over 95 % tested by HPLC. GST-VNP was then digested by enterokinase and removed from GST tag protein. Pure recombinant protein VNP was eventually obtained through centrifugal ultrafiltration, and the total yield was 20 mg recombinant VNP per liter of fermentation broth. Mass spectrometry illustrated that the molecular weight of recombinant VNP was 2864.69 Da. Western blotting and N-terminal amino acid sequencing were further introduced to certify the correctness of VNP.The activity of recombinant VNP was detected by two sets of both cell and animal models. As to animal model, we introduced the isolated rat arterial ring method and the bladder urine collection method to determine the activity of recombinant VNP: In the expansion of vascular aspect ANP