Prokaryotic Expression Of Rat Beta-Defensin-9and Its Polyclonal Antibody Preparation

P-defensin is a family of cationic antibiotic peptides with broad antibacterial spectrum discovered in recent years. This study proposed a process of recombinant expression of soluble rat P-defensins-9. E.coli is adopted as the host cell. A process of recombinant expression of soluble β-defensins-9was proposed, which includes the cloning and the codon optimization of the gene, the construction of expression vectors, the optimization and scaling up of the expression, the purification and bioactivity determination of the product and the obatined of the high quanlity of polyclonal antibody from the rabbit with fusion protein. The main methods and results were as follows.1. The cultivation conditions of the codon-optimized stain of mouse β-defensins-9, BL21(DE3)/pET-32a(+)-rDefb9were optimized considering the induction temperature, induction concentration, induction timing and induction time in shake flask. The optimized culture was:the optimal induction temperature was28℃, the optimal IPTG concentration was0.8mmol·L-1, the optimal beginning induction timing was mid-exponential growth phase, and the optimal induction time was3hours.2. The isolation systems of β-defensins-9from recombined mouse P-defensins-9(rDefb-9) expression system were selected after a process of Ni affinity chromatography, formic acid digestion and freeze drying.3. The rDefb-9polyclonal antibody was obtained from the immune rabbit with the expression fusion protein. The enzyme-linked immunosorbent assay (ELISA) was used to detecte the serum rDefb-9, which laid the foundation for the further study of rDefb-9function.