Cloning And Fusion Expression Of Drosophila Andropin Gene In E.Coli

Based on former's research about the relationship between structure and activity of antibacterial peptides, we designed and synthesized the Drosophila antibacterial peptide─Andropin, which has 34 amino acids.The synthesized whole gene was cloned into the T-easy vector. Choosing an efficient expressing vector─PET32a, we cloned the target gene into it. The recombinant plasmid was transferred into an E.coli host OrigammiB. first the expressed recombinant protein was purified by His-tag purification kit, then digested by enterokinase, and finally its antibacterial activity was tested by agar diffusion assay. 1. In this study, we synthesized two gene fragments which have a short complement sequence and two primers. Then obtained the whole gene by PCR method. When we designed the target gene DNA, we added an enterokinase site and two restrict enzyme sites just before the Andropin gene. This would be a convenient way to choose an expression vector and purify protein. 2. In this study, we framed the recombinant expression vector PET32a-Anp. transformed OrigmmiB and primary identified the positive transformation clone by PCR, then the result of the ligases was confirmed by enzyme digesting and sequencing. After the correct recombinant vector was transformed into the E.coli host OrigammiB, we designed tests with avariety induced temperature and time conditions, in order to improve the amount of the product and obtain the best induced parameters. The Andropin was expressed as a fusion protein in a soluble form with a theoretical molecular weight of 28KDa. Following induction by 1mM IPTG for 6h at 25C, the amount of the recombinant protein reached 20%─30% of the total proteins. 3. After induction and harvest, the cells were disrupted by sonication, the soluble fractions were subjected to affinity purification, and then analyzed by SDS-PAGE, a main band appeared at approximately the 28KD. The fusion protein that was purified by His-tag purification kit was dialysed against 10 volumes of enterokinase digestion buffer, and then digested by enterokinase. The activity assay demonstrated that the recombinant peptide had active ability against the E.coli and Staphylococcus aureaus . This research provided an useful approach to the production of recombinant antibacterial peptides, and established a good foundation to further the structure-activity relationship, the characteristics of antibacterial peptides as well as the design and development of new and more potent antimicrobial peptides to inhibit microorganisms.