Study On Monopterus Albus Antimicrobial Peptides Separation, Purification And Antimicrobial Ability

Antimicrobial peptides are a recently discovered type of antimicrobial activitypeptides and play an important role in immunity. But related researches on Aqutic havefew reported before.This paper were primarily studied the antimicrobial ability and activityof the antimicrobial peptides crude sample from Monopterus albus liver, intestines,moreover were studied the liver and intestines antimicrobial peptides' purification processand their related characteristic.Three experiments were followed.Experiment 1. Antimicrobial Peptides Raw Extraction and Antimicrobialability of Differ Tissue from Monopterus albusThis experiment was used methods of Tris-HCl extraction on antimicrobial peptidescrude sample from induced with 1×10⁹/ml A.hydrophila Monopterus albus' mucilageglue, blood, skin, liver,spleen,intestines,kidney. Methods of extraction by Tris-HCl andrefrigerated centrifugation,got crude sample. The antibacterial activity of the raw samplecan be detected inhibited zone by A.hydrophila. The result of inhibited zone showed (Fig1)antimicrobial peptides crude sample from Monopterus albus liver, intestines hadantimicrobial ability. The raw sample from liver and intestines can be detected inhibitedzone by A.hydrophila,the diameters were respective 11.83 and 10.82mmExperiment 2. Monopterus albus Antimicrobial Peptides Separation andPurificationThis experiment selected raw sample from Monopterus albus' liver and intestinal tractto separate and purifricate antimicrobial peptide.The first step: Sephadex G-25 gelfiltration(Filtration pole standard was 1.5×50cm) got rid of ion from salt. The second step:Gel filtration chromatography on a Sephadex G-50 were purified antimicrobial peptidesfrom Monopterus albus' liver and intestinal, filtration pole standard was1.5cm×30cm,eluent was 0.04mol/L PB(NaH₂PO₄ and Na₂HPO₄), pH7.2, velocity of flow 0.2ml/min,collection 1ml/tube and 2 peaks collected each of liver and intestinal,antimicrobial peptides came from peak 2 each of them had strongest antimicrobial activityon A.hydrophila.The third step: Ion-xechanfe chromatography on Macro-Prep High Spurified antimicrobial peptides from antimierobial ability peak 2.Filtration pole standardwas 2.5×20cm, gradient elution, eluent was 0.04mol/L PB(A₁: 0.04mol/L NaH₂PO₄, A₂:0.04mol/L Na₂HPO₄, B₁: Degassed water, B₂. 2.0mol/L NaCl), velocity of flow1ml/min,collection 1ml/tube and only one peak collected each.Then antimicrobialactivities being tested, antimicrobial peptides came from Monopterus albus' intestinal andliver had strongest antimicrobial activity on A.hydrophila, Escherichia coli take secondplace, the last was Staphylococcus aureur.The results of SDS-PAGE indicated thatelectrophoresis atlas all was one strip and illuminated antimicrobial peptides had achievedelectrophoresis pure. The molecular weights were about 6500 Da.Experiment 3. Study on the antimicrobial ability and character of Monopterusalbus intestinal antimicrobial peptidesStudy showed that the antimicrobial peptides inhibited the growth of Gram-positiveand Gram-negative bacteria. After the antimicrobial activities being tested, the minimalinhibition concentrations (MIC) of the antimicrobial peptides were tested by means ofmicro-volumes-liquor assay. The MIC on A.hydrophila, Escherichia coli, Salmonelladerby,Staphylococcusaureur, Bacillus subtilis were 0.048 ug/uL,0.195 ug/uL,3.125 ug/uL,12.5ug/uL,50 ug/uL,respectively.Besides,the antimicrobial peptides were provided withfollowing characteristic:(1) the antibacterial peptides had antibacterial activity stabilityafter being bathed at 100℃for 0 rain and 30 min to be test against A. hydrophilarespectively, the clear inhibitory rings were observed respectively;(2) the antibacterialpeptides can frozen and thawed in -80℃for different times, the antibacterial peptides hadantibacterial activity after being frozen and thawed in -80℃for 8 times.