| Atherosclerosis is a chronic,progressive arterial disease,with the main clinical manifestations of lipid deposition in vascular and thus causing blood flow some or all interrupt,eventually leading to stroke,myocardial infarction,and other lifecrisis.In the blood,lipids is required for energy storage and some tissue hormone synthetic ingredients,and the increased amount of fat in the blood will cause blood flow slows down,forming the fatty streaks on the arterial wall gradually,thereby promoting fat and cholesterol deposition and forming small nodules attached to the smooth artery intima.The small nodules grow fibrotic scar tissue,promoting calcium deposition and gradually evolving to chalky solid membrane not be able to get rid of（atherosclerotic plaque）.This layer of permanent membrane will impede the normal expansion and contraction of the arteries,thus slowing down the speed of blood flow in the artery,and forming a blood clot which can prevent or stop the blood flow through the artery gradually.Although the exact cause of the disease has not yet been determined,hypertension,hyperlipidemia,diabetes and smoking history have already been confirmed as some important pathogenic factors.Atherosclerosis major clinical symptoms can be divided into four phases:（1）asymptomatic or occult period,atherosclerotic plaque formed,but there is no organ or tissue impaired;（2）ischemia period,vascular stenosis induces the symptom of organ ischemia;（3）necrosis period,thrombus or occlusion of vessel lumen leads to the symptoms of organ and tissue necrosis;（4）fibrosis period,organ or tissue becomes fibrosis atrophy because of long term ischemia,including plaque hemorrhage,plaque rupture,calcification and aneurysm formation.At present,the disease is mainly based on prevention by avoiding intake of high cholesterol and fat from childhood;in the treatment stage will make a change of lifestyle and diet,medication or surgery and so on,such as reducing the intake of saturated fat and cholesterol,taking lipid-lowering and anti-platelet drugs or the arterial intima precise resection.The earliest theory of pathogenesis of atherosclerosis is proposed by Rudolph Virkow in 1859,’vascular endothelial injury,hemodynamic changes and blood hypercoagulability’.Large number of studies have also shown that atherosclerosis is a chronic vascular inflammatory disease,both lipid hypothesis and chronic endothelial injury hypothesis become the main point of research gradually.The elevated level of LDL in plasma penetrated into the arterial wall gradually,resulting in lipid accumulation within the smooth muscle cells and macrophages,then smooth muscle cells proliferated and migrated to the subintima and intima area.Oxidation of LDL chemoattract monocytes penetrating into the intima,transforming to macrophages and then retarding in the subintima gap,but the scavenger receptor on the surface will be conducive to induce more oxLDL entering to cells,further aggravating the progression of disease.oxLDL also has a cytotoxic effect on endothelial cells and results in dysfunction or loss of function of endothelial cells in the damage area.Because of endothelial loss caused by a variety of mechanisms of endothelial injury,platelets aggregate and adhere to the subendothelial tissue and chemoattract monocytes and T lymphocytes,releasing platelet-derived and monocyte-derived growth factors,inducing the smooth muscle cells penetrate into the intima and proliferate,and then synthesizing connective tissue and proteoglycan,forming the fibrous plaque eventually.Some other cells（macrophages,endothelial cells,arterial smooth muscle cells）can also generate growth factors,which giving rise to the proliferation of smooth muscle cell and extracellular matrix.In recent years,atherosclerosis is defined as an autoimmune diseases with multiple factors and characterized as Th1 immune response.Long-term infection,physical or chemical factors stimulates the body’s immune system against self-antigen,which triggers the generation of autoantibodies or sensitized lymphocytes interacted with self-antigen and resulting in autoimmune damage,all above is the main pathogenesis of autoimmune diseases.Now the main treatment for most autoimmune diseases is looking for related self-antigen and vaccine development,which indicates a new research direction for the treatment of atherosclerosis.oxLDL,HSP60,andβ2GpI had been confirmed as three atherosclerosis related self-antigens in previous studies gradually.According to Jan Nilsson,ApoB-100 could be divided into several 20-peptide fragments,which is considered as LDL unique related protein,and synthesize artificially,biopanning the disease related 20-peptide with patient plasma affinity,positive 20-peptide fragments are attached to carrier protein to immune ApoE-/-mice as antigen to observe the preventive effect,the results reveal that p45（661-680）could reduce the plaque area up to about 70%significantly.Recently vaccine derived from effective 20-peptide has been developed into clinical trials,recombinant human MDA-p45 antibody（BI-204）has completed Phase Ⅰ clinical experiments and achieved good therapeutic effect.Now the research focus of atherosclerosis has transited from pathological mechanisms to immunotherapy stage,cholesteryl ester transfer protein（CETP）,lipoprotein a（Lpa）and lipoprotein lipase（LPL）have also been confirmed to be the possible theraputic targets.Based on the research above,we will continue to find another polypeptide in plasma associated with atherosclerosis to explore the immune mechanism of the disease.In this experiment,we want to screen patient plasma special binding peptide by phage display technology;sequencing the positive phage and verifying the reliance by analyzing the binding activity to patient plasma;looking for nature protein which is most similar to 12-peptide sequence and synthesizing corresponding polypeptide;testing the difference affinity of synthetic polypeptide with patient plasma and normal plasma,selecting most possible disease related polypeptides and are conjugated to the carrier,which will immune animal models to further confirm the preventive effect and disease relation of selected polypeptide.Methods1.Identified the capacity of phage display 12-peptide library,diluted as 10-fold series to 109-1011,titer was determined,counted the number of plaques on the plate,obtained per 10 μl phage plaque forming units（pfu）titer by multipling the dilution fold by plaque number.2.Plasma from atherosclerosis patient was coated in a 96-well ELISA plate,screened three rounds with phage display 12-peptide library,measured the titer of each round respectively and calculated the enrichment of screened positive phage.3.Picked up a blue plaque from a titering plate of the third round and amplified monoclone phage,purified phage single-stranded DNA from the supernatant,ssDNA was sequenced and identified by gel electrophoresis,the remaining supernatant was used to test the affinity of positive phage with patient plasma.4.Plasma from atherosclerosis patient was coated in a 96-well ELISA plate,PBS was also coated as control,test the affinity of positive phage with patient plasma by ELISA to confirm the reliability of biopanning process and the results.5.Analyzed the sequencing results by bioinformation,looked for the most similar nature protein,synthesized polypeptide fragments followed the sequence of most similar protein.6.Synthetic polypeptide was coated in a 96-well ELISA plate,interacted with PBS,normal plasma and patient plasma respectively,verified by ELISA and kept a record of absorbance values at 450 nm,analyzed the difference afinity between normal plasma and patient plasma interacting with synthetic polypeptide by statistics.7.Choose COL6A5 and COL6A6 polypeptide by higher difference affinity with plasma,test dose-affinity respectively to make sure whether the best related polypeptide.8.The highest disease-related polypeptide was composited with mcKLH carrier to enhance immunogenicity by using EDC as crosslinker,measured the protein content of synthetic antigen,and calculated the dose of animal immunization.9.Male ApoE-/-mice were received intra-peritoneal primary immunization with synthetic peptide conjugated to the carrier（n=10）,saline（n=10）or alum alone（n=10）at 6 weeks of age,followed by a booster 3 weeks later,diet was switched to high cholesterol chow 1 week after booster and continued until sacrifice at the age of 25 weeks.Results1.The capacity of phage display 12-peptide library is about 2 × 1011 pfu by phage titering,which is consistent with the content provided.2.After three rounds of biopanning,the ratio of elution and input was calculated and the result showed that phage binding to atherosclerosis patient plasma specifically could be enriched,the degree of enrichment was about 310 folds.3.The single-stranded DNA of positive phage was identificated by gel electrophoresis and the result indicated purification was successful,13 sequences of 12-peptide were obtained from 60 positive phages.4.Absorbance value at 405 nm showed that the positive phages bind to patient plasma significantly and the affinity was higher than normal plasma,the ratio could be more than 5 folds,which demonstrated reliability of screening process and the results.5.According to the similarity between the sequence of 12-peptide and nature protein,we found seven similar protein,such as HPS4,UBX2N,TSC2,ITCH,COL6A5,COL6A6 and TG.6.Statistical analysis of absorbance value at 450 nm,the binding activity of all synthetic polypeptides to patient plasma were higher than normal plasma,but the ratio of two polypeptides derived from COL6A5 and COL6A6 can reach more than 2 folds.7.Dose-affinity test showed that affinity of COL6A5 and patient plasma was dose dependent,which illustrates COL6A5 was the best related polypeptide.8.COL6A5 and COL6A6 synthetic peptide were conjugated to the carrier successfully,and protein concentration determination were 3.47 mg/ml and 3.91 mg/ml respectively.Conclusion1.Atherosclerosis related polypeptide can be obtained by biological screening with phage display technology,positive phages binding to patient plasma specifically can also enrich in a large extent.2.After biopanning and sequencing,polypeptides obtained show a high binding activity to patient plasma,similar nature protein can be found by bioinformatics analysis which prove the reliability of biopanning.3.The affinity of corresponding synthetic polypeptide with patient plasma is higher than normal plasma significantly,and the ratio of COL6A5 and COL6A6 can reach more than 2 folds,and dose affinity test approves COL6A5 may be potential disease antigen or therapeutic targets. |
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