Construction And Biological Characteristics Of The OMP85 And OMP76 Gene Deletion Strains Of Riemerella Anatipestifer

Riemerella anatipestifer disease is an acute,contact and septic infectious disease caused by Riemerella anatipestifer（RA）,it mainly infected with domestic ducks,geese,turkeys and other poultry.The main clinical features of this disease are fibrinous pericarditis,perihepatitis,air sacculitis and meningitis.It is one of the most serious bacterial infectious diseases that endanger the duck industry.Diseased ducks have slow growth,decreased quality and high mortality,which have caused serious economic losses to the duck industry in my country.It is indispensable to study the pathogenic and immune mechanisms of RA.Outer membrane protein（OMP）is a protein that exists in the outer membrane of Gram-negative bacteria.Bacteria use OMP to block the bactericidal effect of antibodies or complement and resist phagocytosis for immune evasion.Vitronectin（VTN）is a multifunctional glycoprotein that regulates complement activity at the level of the membrane attack complex MAC.In the previous study of the interaction between vitronectin and RA outer membrane protein carried out by our group,it was found that OMP85（RAYM＿07109）and OMP76（RAYM＿01395）interact with VTN.Therefore,this study conducted further research on the functions of OMP85 and OMP76 genes,and used Mariner transposon insertion mutagenesis to construct a mutant library of RA-YM strain,and explored the construction conditions of the mutant library.The results of the study are as follows:1. Construction of Riemerella anatipestifer OMP85 gene deletion strain and study of its biological characteristicsNatural transformation method was used to construct the OMP85 gene-deficient strainΔOMP85 and the reverted strain CΔOMP85.There was no significant difference in the biochemical and growth characteristics between the two and the parent strain RA-YM.The results of serum survival showed that the survival rate of theΔOMP85 gene-deleted strain was not significantly different from that of the parent strain RA-YM,indicating that the OMP85 gene was not related to the serum complement of RA.In the adhesion and invasion test to vero cell,the ability of theΔOMP85 gene-deficient strain to adhere to and invade vero cell was significantly lower than that of the parent strain RA-YM（P<0.05）.In the virulence test,the LD₅₀of the parent strain RA-YM and theΔOMP85 gene-deficient strain were 3.16×10⁶ CFU and 1.0×10⁸ CFU,respectively.Compared with the parent strain,the virulence of theΔOMP85 gene-deficient strain was about 30 times lower.In the blood and tissue load determination test,theΔOMP85 gene-deficient strain infected ducklings with24h and 48h blood and tissue load levels decreased to varying degrees compared with the parent strain.It is speculated that the OMP85 gene is related to the virulence of RA and participate in the invasion pathway of RA infecting the body2. Construction of Riemerella anatipestifer OMP76 gene deletion strain and study of its biological characteristicsThe OMP76 gene-deficient strainΔOMP76 and the reverted strain CΔOMP76 were constructed,and there was no significant difference in biochemical and growth characteristics between the two and the parent strain RA-YM.Under the condition of 10%duck serum concentration,the serum survival rate ofΔOMP76 was significantly lower than that of CΔOMP76 and RA-YM（P<0.01）,indicating that the OMP76 gene is related to the serum complement resistance of RA.Compared with the parental strain,the adhesion and invasion ability of the deletion strainΔOMP76 to vero cell was significantly reduced（P<0.01）.In the virulence test,the results showed that the LD₅₀of the parental strain RA-YM and theΔOMP76 gene-deficient strain were 3.16×10⁶ CFU and 5.62×10⁸ CFU,respectively.Compared with the parental strain,the virulence of theΔOMP76 gene-deficient strain was about 177 times lower.Comparing the bacterial load of the parent strain and the deleted strain,the bacterial load of blood and tissues of the ducklings infected with theΔOMP76 gene deletion strain at 24h and 48h decreased to different degrees compared with the parent strain.It is speculated that OMP76 is a virulence-related factor of RA and participates in RA adhesion and invasion pathways of the infected body.3. Preliminary exploration of constructing RA-YM random mutation library using mariner transposonThe mariner transposable element was used to construct a transposon plasmid,the effects of the three promoters on the transposition efficiency were compared,and the transposition and screening conditions were explored,and p SAM-Spc Pro was determined to be the best transposon.The ratio of donor strain E.coli X7213（p SAM-Spc Pro）to recipient bacteria RA-YM is 1:4,and the optimal concentration of Mg²⁺during the conjugation and transfer process of the two bacteria is 10 m M.After mixing the donor and recipient bacteria,add them to a 0.45μm filter membrane and place them in a 37°C 50m L/L CO₂ incubator for 8 hours.At this time,the conjugation transfer efficiency is the highest.50 mutants are randomly selected for single-point sequencing.The sequencing results are present.In the bimodal situation,the insertion site cannot be accurately located.In summary,this study preliminarily determined that OMP85 and OMP76 are virulence factors with RA.Both OMP85 and OMP76 are involved in the adhesion and invasion of RA-infected organisms.OMP76 mediates RA’s serum complement resistance.The conditions for constructing the RA-YM Mariner random mutation library have been preliminarily determined,which provides a basis for the later study of the gene function of RA from the whole genome level,as well as the pathogenic mechanism of RA and the screening of subunit vaccine target proteins.