Study On The Characteristics Of DegP In Riemerella Anatipestifer

Riemerella anatipestifer(RA)is a bacterial pathogen that can infect turkeys,ducks,geese,and other poultry.In order to study the role of molecular chaperone in the periplasmic of Riemerella anatipestifer,We choose DegP which maintains environmental stability through enzyme activity and molecular chaperone function and involved in protein quality control,as the research target.The main research contents and results are as follows:1.Enzyme activity characteristics of RA DegPWe find DegP in RA or expressed by E.coli expression system can both cut off the N-terminal signal peptide to change its molecular weight from 56 kDa to 53 kDa,which support by the SDS-PAGE and WB result.Using E.coli expression system,we expressed DegP double marked with N-terminal Flag and C-terminal His,and verified DegP cut signal peptide itself.Beta-casein was used to verify the activity of purified DegP in vitro.The test proves that DegP expressed in E.coli has enzyme activity in vitro.The optimum conditions of DegP activity in vitro were explored by using azocasein as substrate.It was found that the optimum temperature of DegP activity is 44°C and the optimum PH is 7.0.The activation of DegP activity did not require divalent cations,but calcium ions and magnesium ions could promote DegP activity,while zinc ions and manganese ions could inhibit DegP activity.The H140,D170 and S249 are the key sites of DegP activity,DegP lost its activity in vitro when these active sites were disabled,the active site in DegP is a standard serine enzyme active site.And the activity of DegP could be inhibited by PMSF.According to the bioinformatics analyze results and the experiment result of the activity of DegP mutants in vitro,we believe DegP is a serine protease.2.The role of DegP in the resistance of RA against harsh environmentsDegP knock out strain and enzyme activity site mutant strains were constructed.The survival rates of knock out strain and mutant strains were significantly lower than those of wild type strains under the conditions of PH 4.0 20 min,PH 10.0 20 min and 2.4 mol/L NaCl 1 h,and there was no significant difference between the survival rates of wild type strain,knock out strain and mutant strains at 42°C.That means DegP can help RA survive in stomach and gut.DegP is involved in RA invasion.As a conclusion,DegP is a serine protease,H140,D170 and S249 are the key activity sites of DegP.DegP cut signal peptide by itself.DegP plays an important role in the resistance of RA to acidic,alkaline,and hypertonic environments.