Functional Identification Of B739?0093 In Riemerella Anatipestifer CH-1 And The Evaluation Of The Immune Protection Rate As A T9ss Family Protein

Riemerella anatipestifer(RA)is a Gram-negative bacterium belonging to the family Flavobacterium.It always infects 2-3 or 8 weeks old ducklings,causing acute or chronic sepsis with serositis as the main symptom.The disease has a high mortality rate which has brought great damage to duck breeding industry.The main purpose of this study was toevaluate whether type 9 secreted protein B739?0093 can be used as a protective antigen.The experimental results are as follows:1.The identification of RA-CH-1 B739?0093 protein as a type 9 secreted proteinSequence analysis showed that the B739?0093 gene of RA-CH-1 strain encodes a typical type 9 secretion system secreted protein.The functional region of B739?0093(B739?0093_T)was amplified by PCR and cloned into the vector p ET32a,then expressed and purified as a 34k Da protein.After immunization of Kunming mice,the specific polyclonal antibodies wereprepared to provide usable materials for protein localization.Construct the mutant strain RA-CH-1?B739?0093,the complement strain RA-CH-1?B739?0093 p LMF03::B739?0093,the type 9 secretion system core gene mutant strain RA-CH-1?T9SS and the complement strain RA-CH-1?T9SS p LMF03::T9SS,then extract the secreted proteins of each strain separately.In western-blot analysis,B739?0093 was located in the secreted protein of RA-CH-1.Further more,B739?0093 was not detect in bacterialprotein and secreted protein of RA-CH-1?B739?0093,and detected in bacterial protein but not in secreted protein of RA-CH-1?T9SS.Above all,it indicate that B739?0093 is a protein secreted by the Riemerella anatipestifer type 9 secretion system.In fluorescence quantitative experiments,the transcription level of B739?0093 was up-regulated in the iron-ion restricted environment and fur mutant strain,which shows that the gene was regulated by Fur protein-mediated iron ion utilization.It is speculated thatB739?0093 may be involved in the utilization of iron ions in Riemerella anatipestifer.However,when adding different sources of iron ions to the iron ion-restricted medium,it was found that the B739?0093 mutant strain did not affect the utilization of iron ions.When infecting ducklings,the the lethality to ducks of RA-CH-1?B739?0093 decreased significantly compared with wild strain.After 24 hours,the bacterial load in various tissues of the ducklings also decreased significantly.In summary,the B739?0093 protein is a virulence factor secreted by the type 9 secretion system and is regulated by iron ions,which can be used as a potential protective antigen.2.Evaluation of the protective effect of B739?0093 recombinant protein as an immunogenThree-day-old ducklings were immunized with recombinant B739?0093 protein,and challenged with 10-fold wild LD50 strain 12 days later.The results showed that,incomparison with the non-immunized group,the protection rate of the immunized group was 50%.