Differential Proteome Analysis In Intestine Tissue Of Fish Infected With Vibrio Mimicus And Identification Of OmpU Interacting Proteins

V.mimicus is a severe aquaculture pathogen that can cause fish ascites disease to the grouper.The study of molecular pathogenesis of V.mimicus from proteomics provides a new mode for the prevention and treatment of ascites disease of fish.Previous studies have demonstrated that the intestinal mucosa is the target tissue of the colonization of V.mimicus and also proven Omp U is an important adhesion of the bacteria.However,the interaction of Omp U protein in the intestinal mucosa is not clear and Molecular regulation mechanism of pathogenesis of V.mimicus is also not clear.So,This study research the differential proteomics of Grass Goldfish intestinal mucosa infected with Vibrio mimicus by using proteomics techniques to lay the foundation for revealing the molecular pathogenesis of Vibrio mimicus and researching and developing new type of antibacterial agents.This paper has carried on the following research work:1 Infection model establishment of Carassius auratusThe Grass Goldfish infected with the concentration of 1.15×1010CFU/m L V.mimicus isolated strains of bacteria by immersion and intraperitoneal injection,It turns out that experimental fish began to disease and died by intraperitoneal injection after 12 h while the experimental fish not all died by immersion infection,so the optimal infection route is intraperitoneal injection.Experimental fish infected with 4 different doses of bacterial(3.45×109CFU/tail,2.32×109CFU/tail,1.75×109CFU/ tail and 1.14×109CFU/ tail)by intraperitoneal injection,It turns out that experimental fish began to disease and then all died with the 3.45×109CFU V.mimicus in 12 h while other groups of experimental fish are not all dead.so the minimum lethal dose is 3.45×109CFU/tai by intraperitoneal injection with V.mimicus.2 Extraction method determination of proteins in intestinal mucosaNP-40 lysis method,NP-40 lysis method with protease inhibitorand and acetone precipitation method were used to extract the total protein of intestinal mucosa,Extraction effect was evaluated by SDS-PAGE analysis and protein concentration determination.It turns out that the protein concentrations obtain from the three extraction methods are10.05mg/m L,11.81mg/m L and 1.71mg/m L,The protein concentration is the highest and the bands are the most of the NP-40 lysis method with protease inhibitor which is the best method for the extraction of intestinal mucosal tissue protein.3 Differential proteome analysis in intestinal mucosa of fish before and after infectionThe Grass Goldfish infected with minimum lethal dose of V.mimicus by intraperitoneal injection and then 3 Biological repeats normal and agonal intestine mucosa Were taken at each time point.Intestinal mucosal tissue protein was extracted by NP-40 with protease inhibitor and then 2-D gel map analysis by PDQuest8.0 following intestinal mucosal tissue protein was isolated by 2-D electrophoresis.It turns out that a total of 12 protein spots were significantly changed before and after infection,6 protein spots which were up to 16 times in the expression level,and the oher protein spots were down regulated by 4 times.12 protein spots were identified by MOLDI-TOF-MS MS,they are myosin,serum transferrin,transferrin variants A1,alpha actin,load fat apolipoprotein A-I,peroxidase,amylase,protein disulfide isomerase precursor,transferrin variants A,beta actin,copper zinc superoxide dismutase enzymes and gastrotropin.GO functional annotation analysis,Pathway analysis and STRING analysis showed that these proteins play an important role in the formation,immune,antioxidant and energy metabolism of the cell structure,they relate to each other directly or indirectly which involved in 9 biological processes and 9signal transduction pathways.4 Screening and identification of Omp U interacting proteinsHis-Omp U fusion protein was prepared by the conventional method of protein expression and purification.Omp U interacting protein was screen in intestinal mucosa tissue by using His-tag pull down kit with His-Omp U protein as bait protein and intestinal mucosal tissue protein as prey protein.SDS-PAGE analysis show that 5 distinct protein bands were observed in the Pull down group which compared with the control group of bait protein and prey protein.5 differential protein bands are Filamin A,?-actin 2,Calponin-2,?-actinin4 and Transgelin-2 identificated by mass spectrometry.Go functional annotation analysis show that 5 interacting proteins are divided into two types,one is cytoskeletal protein(?-actin 2),another is cytoskeleton regulatory protein(Filamin A,Calponin-2,?-actinin 4and Transgelin-2).Besides participating in many important cellular activities,they are play an important role in the adhesion,internalization and pathogenesis of pathogenic bacteria.In conclusion,this study proved the expression pattern of the differential proteome in intestinal mucosa tissue of fish infected with V.mimicus and preliminary screen interaction protein of Omp U.It laid the foundation to reveal the molecular mechanism of pathogenic V.mimicus and develop a new type of antimicrobial botanicals.