| Sepsis is a systemic inflammatory response syndrome arising from infection caused by pathogenic microorganisms(usually bacteria) or their toxin invading the body.It is a kind of the critical complications related to serious infection,badly wound and bum,big surgery,shock and so on.Sepsis is part of a spectrum of conditions ranging from the acute respiratory distress syndrome(ARDS),systemic inflammatory response syndrome(SIRS) to septic shock and multiple organ dysfunction syndrome(MODS).Sepsis and its complications continue to be the main causes leading to high morbidity and mortality in the intensive care unit(ICU).The mortality associated with these conditions ranges from around 26%in patients with SIRS to around 82% in patients with septic shock.Currently,septis is considered as high morbidity,high mortality and high treat expense,which has been a great burden to economic development and threaten to human health.As estimated,the morbidity of sepsis is about 0.3%.The total number of sepsis cases exceeds 4 million increasing by 1.5% each year.The mean mortality is near 40%.In the U.S.A.,about 750,000 people die of septic shock per year and the morbidity is over 50%.Research about sepsis and its mechanism have been a popular area these years, which draws attention of clinical doctors and scientific researchers.Recently,some successes have been achieved in the study of mechanism and clinical signification, but the clinical therapy is still dissatisfactory.Although people have taken great efforts,the clinical therapy remains an area without significant improvement because of its complicated mechanism.In this modern society,many diseases have been cured effectively,while sepsis is still rampant.All the facts suggest that the basic mechanism of sepsis remains an area that needs intense research and the new method of therapy is worthy of exploring. As is revealed by the epidemiology,about 45%to 60%sepsis is caused by Gram-negative bacteria.Lippolysaccharide(LPS) or endtoxin,is the major component of Gram-negative bacteria's cellwall.LPS is a potent activator of the inflammatory cells such as monocytes and macrophages,which induces those cells to change their constructions and functions,accompanying with the change of producing and secreting.Those performances contribute to the systemic changes observed in gram-negative sepsis or septic shock.Compared with natural cells,LPS stimulated monocytes or endothelial cells expose their active sites of cell surface molecules more densely,which may be closely related to the development of sepsis and can act as potential therapeutic targets.As these changed molecules may play as binding sites on the cell surface for some critical cytokines in inflammation cascade reaction, specific binding peptides of these molecules which can competitively inhibit the cytokines binding to cells,may have potential abilities to block biological effects of the cytokines,such as the activation of the target cell to release more cytokine, accommodating the adhesion and so on.The biologically active peptides that target to specific cells show properties of high performance with low side effects.The key step is to find peptides that specifically bind to monocytes/eddothelial cell stimulated by LPS.Phage-display technology is an effective molecular biologic tool that has been developed and widely used since the 1990s.It describes a selection technique in which a peptide or protein turns into fusion protein with a coat protein of a bacteriophage. The fused protein will be displayed on the surface of the bacteriophage,while the DNA encoding it resides within the virion.One of the most significant feature of phage-display technology is that it links phenotype with genotype successfully. According to this,the expression of a particular phenotype on phage is directly associated with the genetic information contained within the phage particle.The expression of the desired phenotype can be achieved by incorporating the relevant genetic material into the phage genome.Phage display has been used to create a directly physical linkage between a vast library of random peptide sequences to the DNA encoding each sequence,allowing rapid identification of peptide ligands for a variety of target molecules(antibodies,enzymes,cell-surface receptors,etc.) by an in vitro selection process,which is called panning.The simplest panning form is as follows:first,we incubate a library of phage-displayed peptides with plates(or beads) coated with targets,then wash away the unbound phage,and elute the specifically-bound phage.The eluted phage will be amplified before taken through additional binding/amplification cycles to enrich the binding sequences.After 3 to 4 rounds,individual clones will be characterized by DNA sequencing.Random peptide libraries displayed on phage can be used in a number of applications,including epitope mapping,mapping protein-protein contacts,and identification of peptide mimics of non-peptide ligands.The Ph.D.-C7C Phage Display Peptide Library is based on a combinatorial library of random peptide 7-mers fused to a minor coat protein(pⅢ) of M13 phage. The randomized sequence is flanked by a pair of cysteine residues.Under nonreducing conditions the cysteines will form a disulfide cross-link spontaneously, and result in the cyclization of displayed peptides contrasted to the linear peptides. Disulfide-constrained peptide libraries have been proven to be useful in identification of structural epitopes,mirror-image ligands for D-amino acid targets,and development of peptide-based therapeutics.The in vivo phage display technique,which is developed during recent years,is an effective method to fred specific peptide binding to organs or tissues.This method can help us to find the target and confirm the domain in the natural environment—tissues or organs,by using the different character of antigen of the phage.In order to provide a new method and clue for the therapy and mechanism of septic shock,we will screen the specific peptides binding to endothelial cell of liver vasculture of septic shock mouse using the in vivo phage display technique.We injected LPS into four BALB/c mouse from abdomen with dosage of 35mg/kg.All of the mice are male and eight weeks old.After arteria carotis intubatton,,we monitord the blood pressure of the mouse.Septic shock mouse model was established by this way.Then we took four rounds of screening to endothelial cell of liver vasculture of septic shock mouse.The peptide library was injected into normal mouse,in order to subtract the peptides that bind to endothelial cells of liver vasculture of normal mouse.The phage clones were gathered and validated by vivo experiment.15 of 20 phage clones were checked to be positive clones.(The phage recovered from liver is three times higher than control organs).We selected the phage clones at random and amplified the objective segment by PCR method.All the segments were sequenced and transformed into amino acid sequence.In total,we have obtained 1063 right objective segments.Some of the tripeptide fragments included in these heptapeptide sequences were mimotope of functional proteins,and may act as ligands of biochemical recognition units in protein-protein interactions.In this study,shuffling algorithm and completely rearranged peptides were used to calculate the abundance and significance of the tripeptides.After calculation and statistics analysis,we got 3,390 distinct tripeptides and for 200 of them,the appearance of frequency is significant.Then multiple sequence alignment of tripeptides with significance was conducted with Clustal W program,from which we got 62 mimetic peptides considering the properties of amino acid.The mimetic peptides were aligned to mice protein sequence library(SWISS-PROT) through online BLASTP service and mice proteins information containing these mimetic peptides were obtained.Signal peptide predicting program SignalP 3.0 and transmembrane helices predicting program TMHMM 2.0 were used to identify secreted proteins and transmembrane proteins respectively.With the bioinformatics analysis mentioned above,we could acquire objective protein.We validated the phage(LTTWAPA) whose frequency is the highest in vivo experiment and immunohistochemical staining.The LTTWAPA phage recovered from liver was 8.0×10~8/g,which was 40 times higher than the amount recovered from kidney and 80 times higher than brain.While in normal mice,the objective phage recovered from liver was as many as recovered from kidney and brain,all of which were highly lower than the amount recovered from setic shock liver.The control phage recovered from liver,kidney,and brain were nearly the same among septic shock mice.So we drew an elementary conclution:the LTTWAPA phage which we got had a good effect on targeting to liver of septic shock mice.As shown in immunohistochemical staining,the LTTWAPA phage bond to the vasculture endothelial cell of septic shock mice liver,while there was no apparent staining in kidney and brain.As for normal mice,there was no positive staining in all the organs,including liver,kidney and brain.Meanwhile,we set the same amount of M13 phage without inserted sequence as control.The control phage was stained in neither septic shock nor normal mice liver.These data proved that the LTTWAPA phage can effectively target to vasculture of septic shock mousee liver,while not to other organs.On the whole,we have screened vasculture endothelial cell of septic mice livers for four rounds by in vivo phage display technique without knowing the target.Then we subtracted the peptide library in normal mice and got some phage-displayed peptides that specifically bind to vasculture endothelial cells of septic mice livers. Functional proteins and their mimetic peptides were acquired by bioinformatics analysis and prediction and identified to be biologic activity.Taken together,we drew the following conclusions: 1.We successfully established septic shock mice model.2.We screened the peptide library specifically binding to vasculture endothelial cell of septic mice liver.After four rounds of screening,the phage libraries successfully gathered in septic shock mice liver.3.We scraped the phage clones at random and found 15 of 20 clones were positive clones in the vivo experiment.4.We obtained 1,063 peptide sequence by PCR method.We got 3,390 distinct tripeptides,among 200 of which the appearance of frequency is significant through bioinformatics analysis.5.Multiple sequence alignment of tripeptides with significance were conducted with Clustal W program,considering the properties of amino acid,we got 62 mimetic peptides.6.The mimetic peptides were aligned to mice protein sequence library(SWISS-PROT) by BLASTP program and 2,922 mice proteins containing these mimetic peptides were obtained.Signal peptide predicting program SignalP 3.0 and transmembrane helices predicting program TMHMM 2.0 were used to identify secreted proteins and transmembrane proteins respectively.After calculation,98 of them were secreted protein and 226 of them were transmembrane protein.7.We validated the LTTWAPA phage whose frequency is the highest in the vivo experiment and found this phage clone can target to septic shock mice liver effectively.8.Immunohistochemical staining result also proved that the LTTWAPA phage can specifically bind to the vasculture endothelial cell of septic shock mice liver.In summary,we have successfully established a high-performance method to screen peptides that bind specifically to the membrane of endothelial cells.The method includes raw data acquiring through biological experiment,computational analysis of biological data and biology experiment identification of results predicted by bioinformatics. |
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