Siteª²directed mutation and prokaryotic expression vector construction and fusion protein expression of human betaª²defensin 3

    JIN Lin, HAN Yueª²Wu

    Department of Biochemistry and Molecular Biology, Basic Medical College, Lanzhou University, Lanzhou 730000, China

    ¡¾Abstract¡¿ AIM: Siteª²directed mutation of human ¦Âª²defensinª²3 gene was conducted by PCR protocol and the mutated gene was subcloned into prokaryotic expression vector. METHODS: A twoª²step polymerase chain reaction (PCR) was used for the siteª²directed mutagenesis. Two sets of primers (P1, P2, P3, P4) were designed according to human ¦Âª²defensinª²3 gene sequence and the mismatch was introduced into P2 and P3. Mutagenesis was performed in a twoª²step PCR and the amplified fragments from the second PCR, which contain the mutation site, were subcloned into the vector pGEXª²4Tª²1. Recombinant pGEXª²4Tª²1 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 ¦Âª²defensinª²3 mutant. SDSª²PAGE profile showed a clear protein band with a relative molecular weight of 31 000. CONCLUSION: Human ¦Âª²defensinª²3 gene is successfully mutated and expressed, which will help the preparation of antimicrobial peptide. ......