Isolation,identification And Proteomic Analysis Of Mixed Extracellular Vesicles From Cow Rumen

Understanding rumen microbes and their interactions with host animals has been the key to understanding and elucidating ruminant digestive physiology and regulating ruminant production and health.Although studies based on in vitro pure culture and omics techniques have provided important information on the rumen microbial population structure,metabolites,and metabolic pathways,there is still a lack of effective exploration of the interactions between rumen microorganisms and their hosts.tools and means.Extracellular vesicles(EVs)are a group of spherical or cup-packed nano-membrane structures secreted by prokaryotic and eukaryotic cells to the outside of the cell.The important role,which is considered to be the key to the communication and physiological interaction between microorganisms and their hosts,is becoming a new medium and new means to analyze the relationship between microorganisms and hosts.The research based on rumen EVs is very likely to provide new means and approaches for the in-depth understanding of the rumen micro-ecosystem and the digestive physiology of ruminants.However,there is no relevant research and report on rumen EVs.How should EVs in the rumen system be? separation,its particle size distribution,composition and origin,etc.,are still in an unknown state.Therefore,this study was first based on understanding the basic physical and chemical properties of rumen samples,by comparing different high-speed centrifugation times(30,60 and 90 min),different protein removal reagents(ammonium sulfate,metaphosphoric acid and ferric chloride)and different filtration devices(The effect of ordinary and vertical crossflow suction filtration)on the treatment effect of rumen fluid samples to establish a suitable sample treatment method before separation of rumen mixed EVs.On this basis,the separation,purification and characterization of rumen mixed EVs were carried out by combining ultracentrifugation and iodixanol density gradient centrifugation.Preliminary exploration of the source of rumen mixed EVs.The study yielded the following results:(1)After the rumen sample used in the test was filtered through four layers of gauze,the obtained rumen fluid was green and turbid,with a p H value of 7.03,a viscosity of 6.1±0.55mm2/s2,an OD600 of 3.37±0.06,and a protein concentration of 4.02±0.05 μg/μL,the particles are distributed between 800 and 5000 nm,indicating that the rumen sample is a turbid liquid with high viscosity,high protein and rich in large particle components.(2)High-speed centrifugation of rumen samples for 30,60,and 90 min can significantly reduce the large particles above 1000 nm(P<0.05)and the protein concentration(P<0.05),but the effect of different centrifugation time on the precipitation of particles and protein concentration were not significantly affected(P>0.05);high-speed centrifugation significantly reduced the viscosity of rumen fluid(P<0.05)and significantly improved the clarity of rumen fluid(P<0.05),but there was no significant difference between 60 min and90 min(P<0.05).P>0.05);Ammonium sulfate,metaphosphoric acid,and ferric chloride could significantly reduce the protein concentration in the supernatant after high-speed centrifugation of rumen fluid(P<0.05).The improvement of the clarity of the supernatant and the decrease of the viscosity were more significant(P<0.05),but both of these two reagents would cause the dispersion of the liposome particle size an the concentration decreased significantly(P<0.05).The particle size and concentration of liposomes were not affected(P>0.05);the filtration efficiency of vertical cross-flow suction filtration was significantly higher than that of ordinary suction filtration(P<0.05).(3)The particle size distribution of the isolated rumen mixed EVs ranged from 100 to300 nm.They were spherical under scanning electron microscope and had obvious doublelayer membrane structure under transmission electron microscope.After purification,rumen mixed EVs were mainly enriched in the third,fourth,5 density layers,the corresponding density is 1.127～1.199 g/m L;SDS-PAGE and BCA results show that obvious protein bands and higher protein concentration can be observed in the 3rd,4th,and 5th density layers,and3 enrichment layers The particle size of EVs is about 110 nm,and the relative concentration is much higher than that of other density layers;at the same time,the kurtosis of EVs particle size distribution is narrowed,there is no obvious fibrous impurities in the background of electron microscope,and the lipid-to-protein ratio is significantly improved.(P< 0.05).(4)A total of 24,905 peptide maps were obtained through protein profiles,which matched 151,35,5,9,151,35,5,9,151,35,5,9,14,96 and 50 specific peptide maps identified 50,29,5,8,13,91 and 42 proteins respectively.Although the matching amount is not large,it preliminarily indicates that the rumen EVs isolated in the experiment are bovine,bacterial,a mixture of complex EVs derived from fungi,protozoa,archaea,and feed.In conclusion,this experiment established a rumen fluid sample pretreatment scheme with 60 min high-speed centrifugation,ammonium sulfate protein removal and vertical cross-flow suction filtration as the core,and successfully isolated and purified rumen mixed EVs with high purity.Characterization was performed to initially identify rumen EVs as a complex mixture of animal,microbial,and feed-derived EVs.