Design And Expression Of The Hybrid Antimicrobial Peptede PM23 And Studies On Preparing Hemin From Procine Blood

(I)Antimicrobial peptides (AMP), a class of short proteins with a broad spectrum of antibacterial activities, are isolated from a wide variety of organisms including animals, plants and bacteria. They are a key component of the innate immune response in most multicellular organisms. Due to their potent and broad-spectrum antibacterial activities and rare development of drug resistant-bacteria strains, these peptides are of great clinical significance.Pleurocidin, isolated from skin mucous secretion of the winter flounder, displays a strong antimicrobial activity and appears to play a role in the innate houst defence. Misgurin was isolated from the loach, Misgurnus anguillicardatus, and exhibits antmicrobial activities against bacteria and fungi without signifieant hemolytic activity. Hybrid peptides were designed from them. The most potential hybrid peptide was screemed based on the analysis of the structure-activity relationship of antimicrobial peptides using bioinformatics tools. Physical-chemical parameters of hybrid peptides were calculated. The secondary structures of them were predicted and the parameters were optimized. Among various hybrid peptides, pleurocidin(1-8)-misgurin(7-21),abbreviated as PM23, is the most potential one with aα-helix structure and the best cationic and amphipathic property.Based on the amino-acid sequence of PM23, the nucleic acid sequence was designed, synthesized chemically and inserted into a fusion expression vector pGEX-4T-2. The recombinant plasmid was transfered into E.coli BL21 and the expression of the fusion protein was induced by IPTG of 0.8mM. The resulting expression product was assayed with the SDS-PAGE electrophoresis and Western blot methods. At 4 hours of incubation with IPTG under 25℃, the engineering bacteria produced maxium recombinant protein, reaching an amount of the 20% of total bacteria proteins. The fusion protein was purified with GSTrap FF affinity chromatography and cleaved with enterokinse. A main band with an approximate molecular weight of 29KD appeared, suggesting that the fusion protein contains the hybrid peptide. Finally, the purified fusion protein appears antibacterial activity with method of agar diffusion.This research provides an useful method to produce recombinant antibacterial peptides and establishes a solid foundation for the development of new and potent antimicrobial peptides.(Ⅱ)Heme is a prosthetic group in a relatively large number of cellular hemoproteins that carry out diverse biological functions and has been used widely. The investigation on the production of hemin from porcine blood can not only converts the waste to the usefulness, but also prevents the harm caused by porcine blood immediately ejected into circumstance. Therefore, the investigation is greatly ecologically meaningful and economically valuable. The ultrasonic wave cell break method combined with the acetone distillation method was carried on for the extraction of hemin from poricne blood.In order to find the optimum parameters of ultrasonic comminuting red blood cells, the concentration of Hemoglobin as an index and the orthogonal test were adopted to investigate the influences of volume of distilled water, ultrasonic time and ultrasonic power. AHD-577 method was used to determine the concentration of Hemoglobin solution. The results showed that the 1:5(V:V) radio of the distilled water against blood corpuscle and 10 min ultrasonic hemolysis at 250W were optimal conditional of ultrasonic comminuting red blood cells.In order to improve the yield of highly pure hemin, our experiment explored the best conditions for extracting hemin by acetone distillation method. The influences of the volume of acetone, pH and stirring speed and time on the purity and yield of hemin were investigated.The results showed that acetone and red blood corpuscle at a volume ratio of 5:1, a pH value of 2.0 and stirring at 500rpm for 10min were the best conditions for hemin extraction. Under these conditions, the yield and purity of hemin were about 93.4% and 92.5%, respectively.