| Objective: Separation of maggots antibacterial material after secondary immune,and identify the antibacterial mechanism of antibacterial material.Method:1. The cultivation of maggots(1) The preparation of maggot’s eggs: The wild Lucilia sericata was cultured insterile conditions. The eggs of wild Lucilia sericata were sterilized for next step.(2)Maggot breeding: The eggs were cultured in sterile environment, in theillumination for at least12hours daily, in60%air humidity condiction and at25to27°C.(3)The disinfection of maggot: The maggots were soaked in3.5%formaldehydesaline5min, then in2%hydrogen peroxide for3min, and in5%dilute hydrochloricacid solution for5min at last. It was washed for3times using sterile ddH2O after everystep was finished. The maggot should keep their vitality after process.2. The preparation of bacteria(1) The recovery of bacterium: Some colonies were inoculated in20ml LB brothfrom primary Escherichia coli and Staphylococcus aureus using a sterile inoculatingloop. It was cultured at37℃,120r/min for17h until the broth became to turbidity.The active Escherichia coli and Staphylococcus aureus were stored at4℃.(2) The breeding of bacteria: The active Escherichia coli and Staphylococcusaureus (100ul) were added to10ml LB broth at37℃,120r/min for4h. The densityreached to a concentration of108cfu/ml (OD value of0.5) by dilution with a sterile LBbroth.3. The co-cultivation of the maggot with Escherichia coli and StaphylococcusaureusThe co-cultivation of the maggot was divided into eight groups, each group has four maggots, and were cultured for6,12,18,24,30,36,42,48hours withcorresponding bacteria concentration (108-101cfu/ml),. a group which cultured forthe longest time, with the maximum of bacteria concentration and had the mostexuberant vitality was selected as expand cultured group.4. The extraction of maggot100maggots’ larvae was added to2×volumes TNE buffer and centrifuged for10minutesat at4℃,12000rpm. The precipitation was added3×volumes TNE bufferafter removed the supernatants. The supernatant, the initiatory extracts, was obtainedafter boiled for2minutes and centrifuged in the above condition.5. The determination of protein concentration using brandford method6. The MTT assay was used to examine the effect of the extracts of maggot thatwas screened on the proliferation of Staphylococcus aureus. The minimal concentrationof the sensitive concentration against Staphylococcus aureus was considered to be theminimum inhibitory concentration (MIC).7. The effect of plasmid replicationPlasmid with5μL and sterile water with15μL were added to eppendorf tube, and thensequentially added various concentrations of maggot extracts. No maggot extract was acontrol. After mixing, incubation at37℃for2hours, Plasmid was detected by agarosegel to analysis the role of maggot extract for plasmid DNA.8. The replication of DNA for E. coli and S. aureusPolymerase chain reaction used indicating the bacterial genomic DNA as template.Various concentrations of maggot extracts were added to the reaction system.no grainmaggot extract was a control, PCR product was analyzed by agarose gel to study thefunction of maggot extracts for Taq DNA polymerase.9. Observation of E. coli and S. aureus ultrastructure:for maggot extractsE. coli and S. aureus ultrastructure were collected after co-cultured withMICconcentratio maggot extracts for12h. The E. coli and S. aureus in the logarithmicgrowth phase was a control. Cells were harvested3000r/minfor10min. They wereresuspended in2.5%glutaraldehyde after washed with sterile saline. They were fixed inosmic acid; concentration gradient of the ethanol dehydration, ultrastructural changes ofthe bacteria was scanned by electron microscope.Results:1.With the MTT assay, the extracts of maggot had significant inhibition on theproliferation of Staphylococcus aureus, and the minimum inhibitory concentration (MIC) was0.5mg/ml.2. The plasmid did not run out the agarose gel when the concentrations of theextract was10mg/ml; when extract concentration was1mg/ml, plasmid stripsignificantly darker.3. When extract concentration was above0.5mg/ml, PCR product from E. coliand Staphylococcus aureus DNA dose not exist by agarose gel electrophoresis.4. The scanning electron microscopy shows E. coli and Staphylococcus aureussurface dispalyed depressions or holes and even rupture after co-cultured with extractantibacterial substances with MIC concentration of maggot extracts for12h.Conclusion:1.The minimum inhibitory concentration (MIC) of extracts of maggot againstStaphylococcus aureus was0.5mg/ml.2.The maggot after the second immunization insect antibacterial substancesinhibit e. coli and s. aureus bacterial plasmid and DNA replication, and destroy bacteriacell membrane structure, so as to achieve the antibacterial effect. |
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