| Human brain natriuretic peptide (hBNP) is an endogenous peptide produced by the heart, and neuroendocrine stimuli increases BNP secretion as a compensated mechanism. BNP is thought to have its biological actions, mainly via the guanylyl cyclase-coupled natriuretic peptide receptor (NPR-A), thereby increasing intracellular concentration of cGMP. BNP has been approved as a therapeutic approach for acutely decompensated congestive heart failure. However, current BNP application is limited due to its rapid clearance, low bioavailability and short termimal half-life, which indicating the modification should be made on hBNP to produce better pharmaceutical ability. Human serum albumin fusion technology was applied in this paper to produce 4 recombinant fusion proteins of rhBNP. The main conclusions are as follows:In order to prolong the pharmacodynamic and pharmacokinetic half-life of BNP, four therapeutic proteins with different fusion method were designed. The fusion genes of hsa-(bnp)2,bnp-hsa,(bnp)2-hsa and (bnp)4-hsa were obtained by gene recombinant technology and cloned into pPIC9K expression vector, which is optimal expressed in Pichia pastoris.pPIC9K expression vectors inserted with fusion genes were transformed into P. pastoris GS115 by electroporation. Four recombinants with the highest production of HSA-(BNP)2, BNP-HSA, (BNP)2-HSA and (BNP)4-HSA were screened and the yields were 156 mg/l, 212 mg/l, 161 mg/l and 47 mg/l, respectively.The optimal fermentation process in the 500-ml conical flask was investigated. 1 ml activated seed lot was cultured in 150 ml YPD at 200 r/min and 30°C for 28 h, then left the flasks stale to let the pichia cells subsidence. After that, the supernant was removed carefully and the pellet was resuspended into 100 ml BMMY with 2% methanol. The 1.5-fold concentrated culture was induced at 200 r/min and 30°C for 72 h, with 2% methanol supplement every 24 h.Two steps of the protein purification process were established. Firstly, the culture supernatant was 20-fold concentrated using an ultrafilltration device with Biomax-10 membrane. Secondly, blue sepharose affinity column was applied to collect proteins with HSA property. The final purity of fusion proteins was above 95% and the recovery was above 56%.A new method for BNP bioactivity analysis in cellular level was established. The BNP bioactivity was positive relevant to the inhibitory degree caused by rhBNP on iNOS and TNFαmRNA level in Raw264.7. Results showed HSA-(BNP)2 has the best BNP bioactivity among four fusion proteins, which was further confirmed by blood-pressure-lowering treatment in vivo.A novel sandwich ELISA was developed to determine the fusion protein of HSA-(BNP)2 in mouse plasma and the detection limit was between 0.625200μg/ml. The ELISA method had been successfully applied to study plasma concentration of HSA-(BNP)2 in mice after intravenous administration. The terminal half-life of HSA-(BNP)2 evaluated was 2.14 h, compared to 3.1 min half-life of BNP monomer in mouse, the terminal half-life of fusion protein had extended more than 40 times.
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