Study On The Antibacterial Mechanism Of Antibacterial Protein Extract Of Stable Fly For Pathogenic Escherichia Coli

The aim of this research was to study the antibacterial effect and the mechanism about antibacterial protein peptide of stable fly on bovine Escherichia coli O1,O2,O8,O78 in vitro.The in vivo study was about the effect on intestinal mucosal barrier on mice that were infected by E.coli O8.To provide a fundamental basis for antibacterial protein peptide of stable fly on bovine E.coli in vivo and in vitro.This research was divided into 5 parts.(1)To extract the antibacterial substance of maggot using ethyl alcohol,ethyl acetate,acetone,chloroform,petroleum ether.The extract method of antibacterial peptide had antibacterial effect.Adopt this method to stable fly.The results showed that the diameter of inhibition zone that the antibacterial protein extract of stable fly on E.coli O8 was 25.55±0.39mg/mL.The concentration of protein that the antibacterial protein extract of stable fly was 0.653±0.021mg/mL.(2)The research showed that the pathogenic E.coli O1,O2,O8 and O78 had a strong pathogenicity,so choose these 4 bacterial strains to take antibacterial experiment in vitro.The results showed that the extracts of maggot and stable fly both had the effects to destroy the cell membrane and the permeability of cell wall on these4 E.colis.To inhibit the growth of bacterial in 24h,the protein substance and ion that bacterial released could be tested,so the content of protein and the electrivity conductivity were increased.In this way to reach a antibacterial effect.These two ectracts had widely antibacterial spectrum.(3)The experiment was divided into the normal control group with normal saline,the negative control group that inject bacterial and normal saline,the positive control group,the high dose of antibacterial protein extract of stable fly,the medium dose of antibacterial protein extract of stable fly,the low dosage of antibacterial protein extract of stable fly in a period of 7 days,twice a day.At the 5th day to give a intraperitoneal injection with 80%MLD.The results of the negative control group compared with normal control group showed that the content of lactice acid bacteria and bifidobacterium in cecum were significantly decreased(P<0.05),the content of enterococcus and enteric bacilli were significantly increased(P<0.05),the intestinal villi length,crypt depth,the villi/crypt(V/C),the muscular layer,lymphocyte and goblet cell were significantly decreased(P<0.05).The medium dosage of stable fly could regulate the structure of intestinal flora and had a better recovery effect on small intestine mechanical barrier of mice,these effects were close to the positive group.(4)The results that negative group compared with the control group showed that the content of IL-2,IL-4,IL-10,IgA,Ig M,IgG,INF-?,T3,T4 and INS in serum were significantly decreased(P<0.05),the content of IL-6 and IL-8 were significantly increased(P<0.05),IL-6,IL-8,TNF-?,ACTH,CORT,GH and blood sugar were significantly increased(P<0.05).The positive control group and the medium dosage of stable fly could improve the content of Immunoglobulin,Anti-inflammatory cytokine in serum of mice that were infected by E.coli O8,decrese the content of proinflammatory cytokine.In this way to regulate these indexes in serum and to improve the stress resistance.The results that negative group compared with the control group showed that the visceral index,the activity of NK and Phagocyte were significantly decreased(P<0.05).The positive control group and the medium dosage of stable fly could improve the visceral index,the activity of NK and Phagocyte significantly(P<0.05).The results that negative group compared with the control group showed that the content of CD3~+,CD4~+,CD19~+and CD4~+/CD8~+were decreased significantly(P<0.05),CD8~+was significantly increased.The antibacterial protein peptide of stable fly could improve the content of CD3~+,CD4~+,CD19~+and CD4~+/CD8~+on the mice that were infected by E.coli,decresed the content of CD8~+.The positive control group and the medium dosage of stable fly could regulate the content lymphatic subgroup of mice,decrease the content of inhibitory T cell to improve the immunological function.The results that negative group compared with the control group showed that the content of IL-2,IL-13,IL-4,IFN-?,SOD and T-AOC in the liquid supernate of mice were significantly decrased(P<0.05)the content of IL-6,TNF-?and 5-OH were significantly incrased(P<0.05).The positive control group and the medium dosage of stable fly could improve the content of IL-2,IL-13,IL-4,IFN-?,SOD and T-AOC significantly(P<0.05)in mice that were infected by E.coli,decrease the content of IL-6,TNF-?and 5-OH significantly(P<0.05).Therefore,the intestinal mucosal barrier could be destroyed by pathogenic E.coli O8,the medium dosage of stable fly had a best effect.(5)To research the biological diversity of bacterial in blind gut on different group that were infected by E.coli through high-throughput pyrophosphate sequencing.The results showed that there were significant differences in diversity of blind gut on different group.The structure of bacterial colony were related to the concentmiceion of antibacterial protein extract from stable fly.