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In Vivo Screening and Characterization of Peptides Specifically Binding to Vasculature of Liver from Mice with Septic Shock

Author: YuanLiHua
Tutor: JiangYong
School: Southern Medical University,
Course: Pathophysiology
Keywords: Phage display Endotoxin shock Endothelial cells In vivo screening Specific binding peptide Bioinformatics
CLC: R459.7
Type: Master's thesis
Year: 2008
Downloads: 16
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Abstract

Sepsis (sepsis) is caused by a variety of pathogenic microorganisms or their toxins, systemic inflammatory response syndrome (systemic inflammatory response syndrome, of SIRS), severe infection, severe trauma and burns, major surgery and critically ill patients with clinical shock one of the serious complications, further development can lead to septic shock (septic shock), acute respiratory distress syndrome (acute respiratory distress syndrome, ARDS) and multiple organ dysfunction syndrome (multiple organ dysfunction syndrome, MODS), etc.. Sepsis and its complications are faced with the thorny problem of Critical Care Medicine, has been the cause of ICU (intensive care unit, ICU) patients with high morbidity and high mortality mainly due to SIRS mortality rate of close to 26%, pus thyrotoxicosis shock of mortality can be as high as 82%. , Sepsis, there is a high prevalence, high mortality, high cost of treatment, three high phenomenon, has been a huge burden constitutes a serious threat to human health and economic development. According to statistics, the prevalence of sepsis was approximately 0.3% of the total population, the total number of cases of more than four million cases occur each year in China, and the ratio of about 1.5% per year growth, mortality averaged 40%; each year in the United States about 75 people septic shock, the fatality rate can reach 50% or more. In recent years, sepsis and pathogenesis research has become one of the very active frontier, increasingly by domestic and foreign majority of clinicians and researchers a high degree of concern. Sepsis pathogenesis and clinical significance have made some progress, but the Phase III clinical trials for treatment of sepsis, MODS and failed to achieve the desired results. Huge despite paying a great deal of effort, but because of the complexity of the pathogenesis of sepsis, clinical treatment difficult, nearly 40 years, its clinical efficacy and prognosis has not been substantially improved, had constituted a serious threat to human health and the economic and social burden. Tremendous development in the life sciences today, sepsis is still so rampant, undoubtedly caused our attention. These facts show that the fundamental incidence of sepsis link and mechanism of action remains to be in-depth study, new ways of intervention worth exploring. Epidemiological analysis showed that sepsis due to bacterial infection, 45 to 60 percent of the Gram-negative bacterial infections. Endotoxin is a structural component of the cell wall of the outer membrane of Gram-negative bacteria, the chemical nature of lipopolysaccharide (lipopolysaccharide, LPS), the main factors causing sepsis. LPS into the blood circulation, to stimulate mononuclear macrophages, endothelial cells and other inflammatory cells, induced structural and functional changes, and lead to the synthesis and secretion of change, leading to some inflammation-related molecules in the cell surface upregulated or exposed to their respective parts of the active structure. Compared to normal cells, the cell surface of stimulated presence molecule having a difference in the number and / or structurally, closely related to the development of these molecules may occur with endotoxin shock, endotoxin shock therapy can serve as a new candidate targets. These cell surface differences molecule there may be some important inflammatory mediators in the inflammatory cascade of binding sites, looking for its specific binding peptide, likely through competitive inhibition of the corresponding combination of inflammatory mediators and effector cells, thereby neutralizing or blocking the biological effects of these inflammatory mediators, such as further activation of target cells to release cytokines mediated cell adhesion, so as to achieve the purpose of blocking or mitigate systemic inflammatory response occurs. On specific cell having the characteristics of the targeting of the biological activity of the therapeutic effect of the peptide is more efficient and fewer side effects. The key steps to discover the LPS-stimulated cells or endothelial cells of the monocyte-macrophage-specific high affinity binding peptides. Phage display technology is a new technology developed and widely used in the 1990s, the principle is the exogenous polypeptide or a protein with the phage capsid protein fusion expression of the fusion protein will be displayed on the phage surface, and the encoding of this the DNA fusants is located within the phage. A basic feature of the phage display technology is the phenotype and genotype effectively linking, i.e. the genotypic information corresponding to the phage surface a specific phenotype of the phage particle for a specific phenotype, simply related genes can be inserted into the phage genome, the phenotype. Phage display technology to make a large number of of random polypeptide their DNA coding sequence to establish a direct link between the various target molecule (antibodies, enzymes, cell surface receptors, etc.) peptide ligands through a process called panning (panning) in vitro selection procedures can be quickly identified. Most simple panning procedure is incubated total tablet of the phage display peptide library with the packet is a target molecule (or beads), the first wash away the unbound phage, and then eluted specific binding phage. Was eluted phage were amplified, and then the next round of binding / amplification cycles to enrich those binding sequence. After three to four rounds of panning by DNA sequencing of each can be combined with cloning qualitative. Random peptide library displayed on phage surface can be applied to many aspects of the research, including the preparation of the antigen epitope maps to study protein - protein interactions and the identification of non-peptide ligand peptide mimics. The bacteriophage display peptide libraries Ph.D.-C7C random heptapeptide fusion M13 phage minor capsid protein (p Ⅲ) built into a combinatorial library. Display random peptide flanked on each side by a cysteine ??(Cys). Under non-reducing conditions, these two cysteine ??spontaneously form a disulfide bond, a polypeptide of the display cyclized. 7 peptide library is limited by disulfide bonds within the ring has been proven to be able to identify the the epitopes structure mirror ligand and development of the D-amino acid target molecules polypeptide-based drugs. In vivo phage display technology developed in recent years, is looking for organizations, effective means of organ-specific binding polypeptide. This method can be provided in the receptor molecule is not yet clear, the receptors naturally occurring in the environment - tissues and organs as ligand, antigen specific phage short peptides find unknown target molecule, and to determine its domain. In this study, using in vivo phage display technology, endotoxic shock mice liver endothelial cells were screened to find its surface molecules specific binding peptides, to provide new ideas for the treatment of endotoxin shock development mechanism occurs and clues. We selected four BALB / c mice, male, 8 weeks old, a dose of 35 mg / kg intraperitoneal injection of LPS, the carotid artery was cannulated to monitor arterial blood pressure in mice, copy endotoxin shock model. Internal state of endotoxin shock mouse liver endothelial cells four screening, then a subtractive screening, and that is to get a specific binding with the shock in mice liver vascular normal mice, while normal small rat liver vascular non-binding phage library. A preliminary in vivo We sieve phage clones reinfusion experiments verify its endotoxic shock in mice targeting verify the total of 20 phage clones, 15 positive clones (unit weight liver reinfusion into the phage is more than three times that of the control organs). Randomly picked phage clones, after PCR amplification of the objective fragment, DNA sequencing, and amino acid sequences deduced according to the insertion sequence outbound source heptapeptide, to obtain the correct sequence of the inserted fragment 1,063 total. These heptapeptide sequence contains a large number of three peptide residue, wherein the tripeptide motif as a ligand in the the biochemical recognition unit (biochemical recognition units), tend to be some of the functional protein analog epitope (mimotope), so look for these mimic epitope and its corresponding protein's function is of great significance. We completely rearrangement heptapeptide shuffling application (shuffle) algorithm (shuffling algorithm) to count a large number of a particular tripeptide heptapeptide phage display (abundance) and its significant, results showed that 1,063 The positive phage display heptad includes 3,390 different three peptide residue, wherein the abundance significant tripeptide 200. Then, using the Clustal W software contains abundance heptapeptide of multiple sequence alignment (multiple sequence alignment) the nature of the analysis, combined with the amino acid having a significantly higher or abundance tripeptide motif in these seven peptides to find The characteristic short peptide motif (analog epitopes), a total of 62 characteristic short peptide motif; adoption of online services BLAST similarity than on analysis, these short peptide motif with the mouse protein sequence database (SWISS-PROT) , get all the rodents known to contain these characteristic peptide protein; application SignalP and TMHMM predicted software further screening of secreted proteins and transmembrane protein, was obtained from the functional proteins. The maximum number of occurrences phage display heptapeptide (LTTWAPA) oriented effects in vivo identification and analysis of immunohistochemical staining. The guide effect in vivo identification experiments, showing LTTWAPA phage recovered in the liver by weight of shock mice units is 8.0 × 10 8 / g, kidney 40-fold (2.0 × 10 7 / g), respectively, the brain tissue 80 times (1.0 × 10 7 / g). In normal mice verification target phage recovered in the liver by weight of units for no significant difference with the brain tissue and kidney, are much lower than the recovery amount of shock in mouse liver. Control group of empty phage shock organs of mice recovery is basically consistent. The preliminary view is that we screened the phage clones endotoxic shock mouse liver good guide. Immunohistochemical staining showed that, the phage express LTTWAPA sequences located in the endotoxin shock mouse liver endothelial cells, brain tissue and kidney of the shock in mice and normal mice liver, kidney, brain, no obvious staining. At the same time, we set up an equal amount of empty phage as a control, the control group empty phage shock mice and normal mice liver showed no staining. We screened the phage can effectively targeting endotoxin shock mouse liver vascular, without binding to other organs of the shock mice, not combined in the organs of normal mice. In summary, this study in the target molecule is unknown, the use of in vivo phage display technology, endotoxic shock mice liver endothelial cells four screening a subtractive screening with normal mice with endotoxin shock mouse liver vascular endothelial cell-specific binding of a short peptide sequence, the application of bioinformatics technology analysis and prediction of these short peptide sequence, and further identification of the predicted functional protein and biological activity of the peptide mimotopes. Through the above research, we have come to the following conclusions: successfully copied endotoxin shock mouse model; 2. Use in vivo phage display technology, screening to endotoxin shock mouse liver vascular-specific binding peptide library. After four rounds of screening, the phage library endotoxic shock mouse liver effective enrichment; 3. Randomly picked 20 phage clones reinfusion in vivo experiments, 15 clones positive phage clones; 4. pick spots and PCR amplification, got 1,063 heptapeptide sequence, bioinformatic analysis found 1,063 displayed heptapeptide included 3,390 different tripeptide residues, which has a significant meaning tripeptide 200 ; 5. applications software Clustal W and combined with amino acid nature, respectively, contain significant tripeptide base sequence of all seven peptide conducted multiple sequence alignment analysis, obtained a 62 with mimotopes peptide nature and functional characteristics of sexual short peptide; 6. Application the BLASTP procedures and mouse protein database contains the above characteristic short peptide motif protein homology alignment analysis of 2,922 a further secreted proteins and transmembrane proteins using SignalP and TMHMM prediction software, which secreted proteins 98, 226 transmembrane proteins, both secreted proteins and transmembrane protein sequence 20; 7. maximum number of occurrences of the phage clones (LTTWAPA) reinfusion in vivo experiments, reinfusion in vivo preliminary experiments confirmed the phage clones can be effectively targeted to endotoxin shock mouse liver; 8. immunohistochemistry experiments further confirmed the heptapeptide phage show LTTWAPA specifically binds to endotoxin shock mouse liver vascular and positioned at the surface of endothelial cells. In this study, the application of molecular biology techniques to obtain the original data, the use of bioinformatics research methods to analyze biological data to predict disease development related peptide characteristic, further biological experiments, is a highly efficient research via.

In Vivo Screening and Characterization of Peptides Specifically Binding to Vasculature of Liver from Mice with Septic Shock

Abstract

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