The Study And Development Of Chimeric Recombinant Protein With Diplex Anti-Pulmonary Fibrosis Genes

Objective: Neutrophil inhibitory factor (NIF) is the undivided antagonist of integrtinβ2. NIF can inhibit the adhesion and recruitment between neutrophil and endothelial cells. It can prevent the infiltration and saturation of WBC. Keratinecyte growth factor (KGF) is a member of fibroblast growth factor's family (FGFs). KGF is expressed predominantly by mesenchymal cells, and it has epithelial specificity to promote the epithelial growth. A lot of investigations have confirmed the two cytokines have important roles in lung development, inflammation, and repair. Aiming at the pathology of pulmonary fibrosis (PF) and acute lung injury (ALI), we utilized the bio-activity of NIF and KGF to construct the chimeric protein--NKM to improve the pulmonary fibrosis patients'survival, and lay a foundation for further investigations. Methods: To obtain the sequence of NKM, we utilized 8×glycin for hinge region to connect NIF with KGF by over-lap PCR. And made use of pET expression vector to express the chimeric protein in BL21 Star(DE3)plysS. After confirmation of sequencing, expression, purification and western-blot, we evaluated the bio-activity of NKM in vitro and in vivo. Results: We successfully constructed the recombinant chimeric gene of NKM, and its expression level is 25% in BL21 Star(DE3)plysS. NKM had immunocompetence by western-blot. We could get NKM with 80% purity by anion exchange resin DEAE. We indicated NKM could inhibit neutrophil adhesion in a dosage-dependent manner in vitro. It also could inhibit fibroblast growth in a dosage-dependent manner in vitro. The animal model research indicated NKM could inhibit WBC increasing in ALI. It could also significantly inhibit the pathological changes of acute pneumonia and fibrosis induced by bleomycin in vivo. Conclusion: We obtained an anti-fibrosis chimeric protein with double functions. It can cure PF and...