Biological Characteristics Of APEC O1 Strain E516 With Iron-Uptake Gene C2515 Or C2516 Deleted And Development Of Monoclonal Antibody Specific To C2515

Avian pathogenic Escherichia coli(APEC),widely spread among poultry,is well-known to cause colibacillosis in chickens,causing significant losses in poultry industry.The ability to uptake iron in the extra-intestinal environment of birds is prerequisite for APEC survival.For adaptation to the low-iron environments,the bacteria have evolved multiple iron acquisition systems to seize iron from the low-iron environments.However,many components of these iron acquisition pathways are still not clear.two putative iron transport genes with 100%homology to the c2515(768 bp)and c2516(1059 bp)genes of uropathogenic E.coli CFT073 were identified in septicemic APEC O1 strain E516.In this study,we constructed the single and double gene deletion mutants,and their biological characteristic and pathogenic traits were explored both in vitro and in vivo.Reverse transcription PCR(RT-PCR)analyses demonstrated that the mutants did not transcribe the target genes.Deletion of the single or double genes of c2515 and c2516 in APEC E516 weakened its ability to produce siderophore.Consistently,the mutants exhibited growth defect under iron-depleted conditions and decreased the intracellular iron levels in relation to that of the wild-type(WT).Cell infection assays showed that the mutants were more easily eliminated by the macrophage.Inactivation of the c2515 and c2516 genes impaired bacterial colonization in chicken tissues,as well as the pathogenicity based on 50%lethal dose determination.Moreover,the expression levels of several iron uptake-related genes were significantly decreased in the double-deletion mutant.In summary,the c2515 and c2516 may in volve in siderophore-mediated iron uptake and participate in the pathogenesis of APEC Ol strain E516.The C2515 protein in APEC E516 strain is encoded by the chromosomal gene c2515.In this study,the recombinant protein expression plasmid pETl lb-c2515-His were constructed,and then the recombinant C2515 was renatured and purified by urea gradient dialysis,and the molecular weight of C2515 were about 28.2 kDa equal to the expected size.BALB/c mice were immunized with the recombinant C2515.The indirect enzyme linked immunosorbent assay(ELISA)was developed,the optimal coating concentration of the antigen was 1.5 ?g/mL.After the immunization,the antibody titer of the immunized mice was determined using ELISA.Then the spleen cells of immunized mice were fused with hybridoma SP2/0 cells.Using limited dilution method,three monoclonal antibodies specific to C2515 were obtained after multiple subclonal screenings,named 1C4,3D6 and 4B9,and identified as IgG2b and IgG1 respectively.All three monoclonal antibodies were confirmed to react with C2515 in Western blot,and confirmed to react with E516 whole bacteria protein while not with E058 or E522.The monoclonal antibodies provides reliable materials for the study of the function of C2515 protein,and also lays a foundation for further research on its role in the iron metabolism of the APEC E516 strain.