Expression,Purification And Antibacterial Activity Of Grass Carp(Ctenopharyngodon Idella) Serum Amyloid A

Serum amyloid A(SAA)is an acute phase protein synthesized by the liver,its serum concentration increases sharply when the body is exposed to pathogenic microbial infections,mechanical trauma and tumor proliferation,and is one of the biomarker molecules of acute inflammation.In recent years,studies have shown that mammalianSAA has a variety of immunological activities,including recruiting internal leukocytes,inhibiting pathogenic microbial activity,activating inflammasomes,regulating macrophage polarization and promoting angiogenesis,however the immune function ofSAA in fish remains largely unknown.In this thesis,the coding sequence of grass carpSAA(gcSAA)was cloned,which contains 366 nucleotides and encodes a protein containing 121 amino acids,and its N-terminal 18 amino acids are signal peptides.Only oneSAA coding sequence exists in the grass carp genome and has similar structural to the homologSAA of mammals and other teleost fishes was revealed by bioinformatics analysis.Then,the expression pattern of gcSAA were investigate by using quantitative real-time PCR.It was found that gcSAA were mainly expressed by immune organs such as head kidney,liver and spleen.And its expression level was significantly increased under bacterial infection conditions.These results suggest that gcSAA as a class of acute-SAA expression features,may directly participate in the body’s immune process against microbial infection.The secreted gcSAA is an amphipathic protein with a predicted molecular weight of 11.79 k Da and the PI of 9.98.It has a positive charge in acidic and neutral environments,and a hydrophobic N-terminal sequence,which resembles the physicochemical characteristics of antimicrobial peptides.Furthermore,in order to investigate the immune function activity ofSAA in grass carp,recombinant grass carpSAA(rgcSAA)were prepared by using Escherichia coli prokaryotic expression system,and the expression efficiency was significantly improved by means of codon optimization,which helped obtain a large amount of rgcSAA.The antibacterial results showed that the proliferation of Aeromonas hydrophila was significantly inhibited and the number of viable bacteria was reduced by rgcSAA.In other words,gcSAA has directly bacteriostatic activity.On the other hand,these bacteriostatic experiments above were also performed on Edwardsiella piscicida,which also caused an increase ofSAA expression in grass carp.Results showed that Edwardsiella piscicida was not inhibited by rgcSAA.In one word,gcSAA showed selectively bacteriostatic activity.To gain further insights into the mode of antibacterial action of gcSAA and the reasons for the selectivity difference,this thesis demonstrated that rgcSAA could directly bind to bacteria,but the propidium iodide uptake test proved that rgcSAA only destroys the integrity of the bacterial membrane of Aeromonas hydrophila,causing the loss of intracellular substances or the influx of external material,and resulting in bacterial death.This perforating effect of gcSAA may be accomplished through its N-terminal hydrophobic sequence,but it has no significant effect on Edwardsiella piscicida.In conclusion,the expression pattern of grass carpSAA was analyzed in this study,the grass carpSAA protein was cloned and expressed,and its directly bacterial inhibitory activity was verified,the possible mechanism of its antibacterial action was preliminarily explored.These results are helpful to reveal the physiological significance of grass carpSAA,and provide ideas for the in-depth study ofSAA function.