Expression Of Bovine Neutrophil β-Defensin 11 In Escherichia Coli And Its Antibacterial Activity Analysis

Bovine neutrophilβ-defensins(BNBDs) include 13 kinds, BNBD1-13. The natural BNBD11 has antibacterial activity against Escherichia coli and Staphylococcus aureus. In this study, we expressed BNBD11 in E. coli and purify it and analyzed the antimicrobial activities of the purified recombinant BNBD11 for the first time. The research is devided into 3 parts:1. Synthesis of the optimized BNBD11 gene and construction the expression vector. Based on the amino acid sequence of natural BNBD11 and E. coli preferred codons, we optimized BNBD11 gene, added cleavage site of formic acid, and artificially synthesized the optimized BNBD11 gene. By the restriction enzyme cutting sites EcoR I and Hind III, the target gene was inserted into the prokaryotic expression vector pET32a, and then transformed the recombinant plasmid into E. coli DH5α. The PCR identification showed a 135bp stripe as expected, and sequencing result was exactly the same as we designed, which proved the expression vector pET32a-BNBD11 had been successfully constructed.2. Expression and purification of the fusion protion. We transformed the recombinant plasmid pET32a-BNBD11 into the E. coli BL21(DE3). The bacteria BL21-pET32a-BNBD11 was induced and then we got the fusion protein Trx-BNBD11 of 21 kD by SDS-PAGE. After the inducing conditions were optimized, the fusion protein was mainly soluble. The fusion protein was purified by Ni Sepharose column affinity chromatography. After dialysed, the fusion protein was cleaved with formic acid by 24h at 50℃.3. Purification and antibacterial activity assay of BNBD11. The mixture of cleavage was purified by RP-HPLC. Antibacterial constituent was screened by antibacterial activity assay. Tricine-SDS-PAGE showed recombinant BNBD11 has high purity. The antibacterial activity of recombinant BNBD11 was assayed by Micro-dilution, and the results showed that recombinant BNBD11 have antibacterial activity against E. coli ATCC25922, and MIC was 20μg/mL. But its antibacterial activity against S. aureus ATCC2592 was not obvious.In this study, we constructed the engineered bacteria with optimized BNBD11 gene and got the high purity recombinant BNBD11. We provided a useful approach to the production of the recombinant antimicrobial peptide with high biological activity and established a good foundation for further study of BNBD11.