The Effect Of Edwardsiella Tarda's Infection On Eel Liver Cells And Study Of The Functions Of Its Infection-related Gene Puah

Edwardsiella tarda is one of the major pathogens of eel.To understand the interactive mechanism of E.tarda and eel in the pathogenesis,Differential proteomic analysis of eel liver cells infected by E.tarda EIB202 were performed and partial functions of E.tarda EIB202 infection-related genes PuAH were studied in this experiment.In one hand,build a infection model of E.tarda EIB202 in vitro,track the Cytopathic and identify the key time points of infection.Extract cell protein in different infection time,analysis them by differential proteomics technology(iTRAQ)and find the expressed differential proteins,then make a KO definition,GO definition and cluster analysis to them.In the other hand,clone and express the infection-related genes PuAH of E.tarda EIB202 by prokaryotic expression technology,The recombinant protein was purified and used to immune New Zealand rabbits to prepare polyclonal antibodies.Then the characterization of expression product and Antisera were analysised through ELISA,Western blotting,Hemolysis test,agglutination test and adhesion test.The results obtained are as follows:The differential proteomic analysis of liver cells in different infection periods:the infection model of E.tarda EIB202 to eel liver cell was constructed successfully,Cells had characteristic lesions in key infection time:cells begin to atrophy and cell gap becomes larger after 2 hours;cells begin to round,swelling,nuclei obviously,almost semi-adherent state after 4 hours;some cells already apoptosis,some blank area Appeared at the bottom affter 6 hours.Through analysising iTRAQ data,12 different proteins were found,of which there were 4 in 2 h group,were up-regulated protein;5 in 4 h group,were also up-regulated protein;8 in 4 h group,7 up-regulated protein and 1 down-regulated protein.These proteins were related with energy metabolism,biosynthesis,sorting and degradation of protein,lipid transport,regulation of apoptosis and so on.And the proteins with similar function had the similar changing trends in different infection time.The verification to the functions of E.tarda EIB202 infection-related genes PuAH:The expression product was fusion protein with 42 ku molecular weight,and had immunogenicity and antigenicity,but no hemolytic to erythrocytes of Eel.Yet the antisera could inhibited the hemolytic of E.tarda EIB202 outer membrane protein to some extent,and could agglutinate E.tarda EIB202.In adhesion test,the fusion protein had no adhesion blocking effect and the abbit antiserum also had no significant inhibition effect to E.tarda EIB202.In summary,this assay describes the changes of the proteins of eel liver cells under the infection conditions of E.tarda EIB202,and Verified that E.tarda EIB202 PuAH gene are associated with agglutination and hemolysis.And provideed reference data for the study of the pathogenic mechanism of Edwardsiella tarda.