Separation, Purification And Gst Fusion Expression In Escherichia Coli Of Antimicrobial Peptides From Pseudosciaena Crocea

The extensive use of antibiotics and chemotherapeutics to control aquaculture diseases has resulted in the development of drug-resistant bacteria, environmental pollution and residues in fish. It has been a hot point to study and exploiture a new antibiotic substitute. The research of more effective and safe means to control aquaculture diseases is increasing with the demand for environment-friendly aquaculture. Antimicrobial peptides(AMP) are short peptides contained antibacterial activity and widely produced by microorganisms, plants, mammals and human et al, and recognized as an essential component in the first line of the host defense system. Furthermore, Antimicrobial peptide has great potential and can be applied in many fields as new therapeutics for their advantages, such as high antimicrobial activities, wide activity spectrum, rapid killing action, and not easy to induce resistant pathogenic strains.The present paper described research of the isolated and purified of antibacterial peptides from gastrointestinal tract of Pseudosciaena crocea by acid extract, solid phase extraction, Sephadex G-25 and RP-HPLC. The results showed there were many substances in gastrointestinal tract of P. crocea which exhibited antibacterial activity. Two isoforms with molecular weights below 5000Da, named AMP-W2 and AMP-W3, were purified from the tissue. Primary analysis of amino acid showed that the amino acid compositions of AMP-W2 and AMP-W3 maybe were Lys, Gly, Ala, His, Val, Leu, Phe, Tyr, Trp,Thr, Ser and Asp, Glu, Gly, Val, Ser, Tyr, Cys, Ala, Thr, Ile, Leu, lys, respectively. The exact molecular weights and sequences of amino acid were worth of further study.The antibacterial activity of the AMPs were detected by agar-diffuse method using of Vibrio alginolyticus as the indicator bacterium. It was found that the minimal inhabit concentrations of the AMP-W2 and AMP-W3 were 5.0μg/ml and 1.25μg/ml, respectively; stronger antimicrobial activities were recorded in sourish condition than in alkaline solution; AMP-W2 and AMP-W3 were hemolytic for human red blood cells when its concentration higher than 2.5μg/ml and 10μg/ml respectively; AMP-W2 and AMP-W3 displayed a broad spectrum against Gram-negative, Gram-positive bacteria and fungi, but they expressed less activity against fungi. The present paper is the first time that isolation and purification from the gastrointestinal tract of fish, and it helpful to a better understanding of the mechanism of antimicrobial and the innate anti-infect defense system of fish.According to the sequence of the cDNA, gene-specific primers were designed and synthesized to amplify the mature peptide sequence from Pseudosciaena crocea. The amplified fragment was inserted into pGEX-4T-3 and the expression plasmid GST-AH was constructed. The plasmid was transferred into BL21 and induced by IPTG at low temperature. The fused protein was induced to be transformed and translated slowly so as to form soluble protein in cells by 0.8mM IPTG at 25℃for 6h. The results of desitometric scan analysis of SDS-PAGE showed that, the cloned protein expressed highly in the form of inclusion body in cells and amounted to about 42% of the total bacterial protein.