| ObjectiveIn this study, measurement of the our group pre artificial design, synthesis, bacteria thehybrid peptide expression CecropinA-thanatin against clinical isolates of Gram-positive,Gram-negative bacteria in vitro antibacterial activity. Our group through animal experiments,different doses of the hybrid peptide, different route of administration on the mice growthperformance and immune function study and discussion, and laid the foundation for theclinical application of genetically engineered antimicrobial peptides.Methods1.The hybrid peptide synthesis to our group of artificial design CecropinA-thanatin study,scale protein expression and expression of the separation of the supernatant, mixed expressionof different batches of the product were measured after the mixture of MIC and clinicalisolates of Gram-positive bacteria （Staphylococcus aureus） and Gram-positivebacteria-negative bacteria （E. coli） Determination of antibacterial activity in vitro;2.Select weeks of age according to the purpose of the experiment suitable strains oflaboratory mice, and packet weighing; processing factors were high, medium and low dosegavage antimicrobial peptides, the middle dose intraperitoneal injection of antimicrobialpeptides, intermediate dose gavage gentamicinprime, the blank control group was given thepPICZαA empty vector transformed yeast supernatants, each processing method is acontinuous treatment for4weeks; dislocation mice were sacrificed after4weeks, weighingthe mass of the body weight, spleen, thymus;3. Intraperitoneal injection of2%SRBC immunized mice, killed the mice, make sure the spleen fresh and sterile then take the spleen lymphatic value-added test and antibody cellsgenerate test;4. One hour prior injection of5%chicken erythrocytes, dislocated mice were sacrificeddraw peritoneal exudate smear microscopy and oil microscope phagocytic rate ofmacrophages, macrophage phagocytosis index;5. Eyeballs to take blood serum was collected and separated in the ELISA test fordetection of serum TNF-α levels; collected mouse intestinal tissue, frozen in liquid nitrogenand placed at-80℃.Take quantitative PCR test for detection of jejunumin TNF-α levels.Results1.Expand the expression of the hybrid peptide CecropinA-thanatin, And Separation,mixing each batch expression products. The MIC was determined that hybrid peptide mixtureto0.45×10-3mol/L. Antibacterial activity was measured, CecropinA-thanatin hybridpeptide has obvious antibacterial activity against Gram-positive bacteria （Staphylococcusaureus）and Gram-negative bacteria （E. coli）;2. By observing the growth performance of the mice in each group, the effect of differentprocessing factors shows that the active, response capability, hair color, weight, growth ratebetween the mice in each group after4weeks is different. High dose CecropinA-thanatinhybrid peptide gavage group mice were significantly better than other groups of mice;3. Index of immune organs of mice in each group were measured, the effect of differentprocessing factors, after4weeks between each group of mice spleen index, thymus index isdifferent, Gavage high-dose CecropinA-thanatin hybrid peptide can significantly improvethe mice spleen index and thymus index;4. After determination of mice spleen lymphocyte proliferation in each group,we foundthat in the role of different processing factors, after4weeks between each group of micespleen lymphocyte stimulation index is different. It can improve the cellular immune functionin mice by gavage and intraperitoneal injection of intermediate dose CecropinA-thanatin of hybrid peptide.5. Detected in each group of mice the number of antibody-producing cells, the role of thedifferent processing factors between the mice in each group, four weeks after theantibody-producing cell number is different, the gavage intermediate doseCecropinA-thanatin of hybrid peptide can improve humoral immune function;6. Macrophage phagocytosis were assayed, and the role of the different processingfactors the macrophage phagocytosis rate between the mice in each group after4weeks,macrophage phagocytic index is different, gavage intermediate dose the heterozygous peptideCecropinA-thanatin can significantly improve the mouse macrophage phagocytosis;7. Testing groups of mice serum, jejunum tissues of TNF alpha protein and mRNAexpression levels, Under the influence of different process factors, between groups of miceafter4weeks of the concentration of TNF alpha and mRNA expression level is distinct,gavage low-dose CecropinA-thanatin hybrid peptide, gentamicin can significantly increasethe concentration of TNF alpha in mice and mRNA expression level;ConclusionsExpress large quantities of synthetic CecropinA-thanatin hybrid peptide and mixture byMIC test determination of the MIC value of0.45x10-3mol/L, in vitro antibacterialexperiments confirmed on clinical isolates of Gram-positive bacteria,Gram-negative bacteriahave antibacterial activity; This study to conduct animal experiments on the basis of thehybrid peptide, verify their biological functions in the body, as well as theCecropinA-thanatin hybrid peptide growth traits and immune function in mice, concludedCecropinA-thanatinhybrid peptide growth performance of mice, can improve the growth rateof body weight in mice; Suitable CecropinA-thanatin hybrid peptide dose and route ofadministration can significantly improve the mice’s immune organ index, cellular, humoralimmunity, macrophage phagocytic capacity. The experimental results show the hybridpeptide CecropinA-thanatin that our group designed, synthesized, and expressed can improve growth performance and immune function of mice. Many of today’s antibiotics produceresistance, the emergence of the hybrid peptide has a huge application potential and it providea new way of thinking for people to find the ideal application.In this study we researched andexplored the vivo applications of the hybrid peptide CecropinA-thanatin as well as the impacton the mice growth performance, immune function, and laid the foundation for the clinicalapplication of the genetic engineering of antimicrobial peptides. |
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