Expression And Characterization Of A Long-Actiing Recombinant Bikunin

Bikunin is a chondroitin sulfate-containing plasma protein synthesized in the liver, and eliminated with urine. In vitro, it can inhibit a large number of proteinases such as trypsin, chymotrypsin, granulocyte elastase,plasmin and also has anti-inflammatory properties at sites of inflammation. Due to its special properties, bikunin has been widely used as a supportive drug for patients with inflammatory disorders such as pancreatitis, acute circulatory failure. Till now, the commercially available bikunin, also referred to as ulinastatin, is mainly derived from human urine. Because of its short half-life,limited source materials security risks and purification difficulties, its production and applications are limited. Therefore, trying to find a way to obtain high inhibitory activity and long half-life bikunin, has been a great concern now.Nanobody is a novel genetically engineered antibody, whose parent antibody exists in the serum of the camel, and is composed of only heavy-chain dimmers.anti-human serum albumin nanobody (aAlb) can be fused to therapeutic proteins and other therapeutic compounds and entities in order to increase the half-life.These findings therefore open new perspectives in the engineering nanobody-based drugs as a flexible platform to increase the half-life of Bikunin.In this work, a novel aAlb-Bikunin fusion protein was expressed in CHO using recombinant DNA technology and purified by Ni2+metal chelating chromatography.In vitro bioactivity was confirmed by trypsin inhibitory activity assay,and the affinity between aAlb-Bikunin and human serum albumin was analyzed using BIAcore.The results demonstrated that the secretory aAlb-Bikunin fusion protein identificated by SDS-PAGE was about40kD.the fusion protein has a significantly trypsin inhibitory activity up to5800IU/mg,The affinity constant with human (mouse) serum albumin was3.93×10-8M and4.86×10-8M analysed by a1:1stoichiometry in SPR. In vivo, blood clearance of aAlb-Bikunin is comparatively longer with a half-life of6h.It is evident that a novel fusion protein with high activity and long half-life has been created to overcome the shortage of extraction and prolong the blood circulation time.So it shows a promising prospect and important application value in the biomedical engineering and disease treatment。...