Site-directed mutation and prokaryotic expression vector construction and fusion protein expression of human beta-defensin 3

AIM: Site-directed mutation of human β-defensin-3 gene was conducted by PCR protocol and the mutated gene was subcloned into prokaryotic expression vector. METHODS: A two-step polymerase chain reaction (PCR) was used for the site-directed mutagenesis. Two sets of primers (P1, P2, P3, P4) were designed according to human β-defensin-3 gene sequence and the mismatch was introduced into P2 and P3. Mutagenesis was performed in a two-step PCR and the amplified fragments from the second PCR, which contain the mutation site, were subcloned into the vector pGEX-4T-1. Recombinant pGEX-4T-1 vectors were transformed into compotent cell BL21. Restriction analysis and PCR were performed to identify the recombinant plasmids containing the DNA fragment of interest, followed by sequencing. RESULTS: We obtained a 138 bp DNA fragment which was identical to human β-defensin-3 mutant. SDS-PAGE profile showed a clear protein band with a relative molecular weight of 31 000. CONCLUSION: Human β-defensin-3 gene is successfully mutated and expressed, which will help the preparation of antimicrobial peptide.