The Purification And Isolation Of Antimicrobial Peptides From The Grass Carp Intestine And The Research On Physicochemical Properties

Antimicrobial peptide is an important part of innate immunity systems, it is the non-specific immunity, can be rapidly synthesized, to resist the proliferation of pathogenic microbes. The speed of its response to specific immune system is unparalleled. So it can play important roles in Prevention and treatment of disease harm to fish and improved varieties. The small intestine of grass carp may possess one or more antimicrobial peptides. The study is that applying SepadexLH-20 size exclusion and RP-HPLC purify the antimicrobial peptide from grass carp. Ulteriorly we research the biological function and physicochemical properties.1. The isolation and purification of antimicrobial peptide from grass carp intestine.After the fresh small intestines of grass carp were treated by grinding, ultrasonic treatment, and 5% acetic acid extraction antimicrobial peptides was isolated from small intestines of grass carp by SephadexLH-20 gel filtration chromatography and RP-HPLC. The fractions obtained were tested against Eseherichia.coli. The fractions showing the antimicrobial activity underwent further characterization. Via changing the condition of the RP-HPLC, the fraction of the first RP-HPLC was purified again, the collection was lyophilized and stored 4℃.2. The research on the antimicrobial activity and the physicochemical propertiesUsing slip diffusion assay, its antimicrobial activity against 7 bacteria strains (pathogenic Escherichia coli,pathogenic Staphyloccocus aureus,Streptococcus,Aeromonas Hydrophila,Myxococcus piscicola,Salmonella,Pasteurella) was researched. The result shows that the intestine antimicrobial peptide was strongly active against all the bacteria strains above Staphyloccocus aureus. The results preliminarily indicated that the small intestine antimicrobial peptide was strongly active against bacteria and possessed broad antimicrobial spectrum.Using different temperatures incubating the intestine antimicrobial peptides 30mins, its antimicrobial activity was tested by Escherichia coli. The result shows that after high temperature incubating, the activity of antimicrobial peptide was not changed. The result revealed that the grass carp intestine antimicrobial peptide had thermal stability.Using trypsin digesting the antimicrobial peptide 16 h, at 37℃, the recovery of the antimicrobial peptide by RP-HPLC was tested antimicrobial activity. The results show that the retention time of the antimicrobial peptide in RP-HPLC system and the antimicrobial activity were not changed. The result indicated that the antimicrobial peptide may be lack of lysine or arginine in primary structure or the antimicrobial peptide had anti-trypsin digesting characteristic.Morphological observation of pathogenic Escherichia coli,pathogenic Staphyloccocus aureus,Aeromonas Hydrophila treated with the small intestine antimicrobial peptide, respectively at 37℃for 30 minutes under scanning electron microscopy (SEM) showed that the surfaces of the tested bacteria above became considerably rough, erimpled, shriveled, distorted and blebbed. The results provided conclusive evidence that the small intestine antimicrobial peptide was membrane active.3. The analysis of the N-residue amnio acid of antimicrobial peptide from grass carpUsing GlycoProTM Enzymatic Deglycosylation Kit treated antimicrobial peptide and recovering the digesting product by RP-HPLC, the product was analyzed MALDI-TOF-MS, the molecular weight was 2762 Da, the product was analyzed by automated Edman degradation sequencing. We obtained a 30 residues amino acid sequence (VKGLVGFAGRRGPPGPRGPVGPAGAKGHQG), this sequence was predicted and analyzed by online bioinformatics database (APDatabase).