Prokaryotic Expression Of The Antimicrobial Peptide2IQ3and Its Mutant,and The Detection Of Its Antifungal Activity

Aims:To construct the expression vector of antimicrobial peptide2IQ3and its mutant by gene engineering technology, purified the fusion protein and evaluate their antibacterial activity in vitro.Methods:The gene sequence of2IQ3was designed according to2IQ3amino acid sequence and the preference of E. coli..The gene of2IQ3was synthesized, then the gene sequence of enterokinase and EcoR I were added to the5’flank and terminal condon TAG and the gene sequence of Hindlllwas added to the3’flank, finally three protective bases were append at the both end. The gene sequence of2IQ3was amplified by PCR, digested by EcoR I and HindⅢ and cloned in to prokaryotic expression vector pET-28a (+) to get the recombinant expression vector pET-28a (+)-2IQ3. The recombinant expression vector pET-28a (+)-2IQ3was transformed into DH5αand selected by kanamycin plate method to obtain the positive recombinant bacteria. It was further confirmed that the positive recombinant bacteria contain the gene sequence of2IQ3by PCR. Using AP database, the mutation of A23T24and A32T33of2IQ3sequence into CA and CA by PCR mutagenesis methods result in alanine instead of aspartic acid at the first and fourth amino acid. The gene of2IQ3m was amplified by PCR when mutant primer P1and P2was design using Premier5.0software and the plasmid of the positive recombinant bacteria mentioned above was used as amplified template. The2IQ3m gene and the expression vector pET-28a (+) were digested and jointed to form the recombinant expression vector pET-28a (+)-2IQ3m. It was transformed into BL21and selected by kanamycin plate method to obtain the positive recombinant bacteria. It was further confirmed that the positive recombinant bacteria contain the gene sequence of2IQ3m by PCR. The fusion protein was produced by IPTG induction and purified by the Nickel affinity chromatography column. The fusion protein was further identified by SDS-PAGE and its concentration was determined by ultraviolet specterphotometry. Antibacterial activity of the fusion protein was evaluated by liquid growth inhibition test in vitro.Results:2IQ3mutant was obtained by designing and modifying the amino acid sequence of2IQ3. The physicochemical properties were analysed by using AP database. We found that the2IQ3mutant was much better than the parental2IQ3in the isoelectric point, positive charge, half-time, lyophobic rate and hydrophilia, suggesting the stronger antibacterial activity of2IQ3mutant. The recombinant expression vector of2IQ3m was carried out by gene engineering technology and further identified by DNA sequence analysis. The result showed that the positive recombinant bacteria include the gene sequence of2IQ3m. The concentration of the fusion protein was0.502mg/ml after the fusion protein was purified by the Nickel affinity chromatography column. The antibacterial test showed that2IQ3mutant can inhibit the growth of Candida albicans, suggesting its antifungal activity.Conclusion:The prokaryotic expression vector of antimicrobial peptide2IQ3and its mutant was successfully constructed and expressed. The fusion protein was purified and observed to have antifungal activity. Hopefully, they can be developed to be novel antifungal drug.