| Avian pathogenic Escherichia coli(APEC)is a type of bacterial pathogen that is parasitic on the intestine and the surfaces of intestinal mucosal,and can cause local or systemic infections.APEC belongs to extraintestinal pathogenic Escherichia coli(ExpEC).It is the primary pathogen causing avian colibacillosis.The wide distribution of APEC has brought great economic losses to the poultry industry around the world.At the same time,APEC poses a serious threat to human food safety.At present,the known virulence factors of APEC include adhesion,endotoxin,outer membrane protein,iron acquisition system,type III secretion system,etc.Outer membrane protease T(OmpT)belongs to the Gram-negative bacteria omptin family.Studies have shown that OmpT plays a role in enhancing the adhesion and invasion ability of bacteria,and is related to the pathogenicity of APEC.Also,OmpT has good immunogenicity,so it has certain research value.The OmpT protein in APEC E05 8 strain is encoded by the chromosomal gene(comp T)and the plasmid gene(pompT).In this study,the recombinant expression strains pET-28a-compT and pET-30a-pompT were resuscitated and identified,and induced by IPTG,and then renatured purified by urea gradient dialysis.The PCR results showed that the gene size of pET-28a-compT and pET-30a-pompT were 894 bp,which were consistent with the expected size.SDS-PAGE results showed that both recombinant proteins were expressed in the form of inclusion bodies.The molecular weight of cOmpT and pOmpT were 36 kD and 33.6 kD,which were consistent with the expected size.The purity of the two recombinant proteins is well.Western blot results showed that the antigenicity of cOmpT was also well.BALB/c mice were immunized with the expressed and the purified cOmpT.An indirect enzyme linked immunosorbent assay(iELISA)was developed,the optimal coating concentration of the antigen was 0.625 ?g/mL and the optimal serum dilution was 1:6400.After the fourth immunization,the antibody titer of the immunized mice was determined using iELISA.The spleen cells of immunized mice were fused with SP2/0 cells.Using hybridoma cell technology and limited dilution method,three monoclonal antibodies specific to cOmpT were obtained after multiple screenings,named 1G8,2C3 and 2G3 respectively,and identified as IgG2b.The titers of monoclonal antibodies in the cell culture supernatant were 1:200,1:3200 and 1:3200 respectively,and the ascites titers of 2C3 and 2G3 were 1:512000 and 1:256000.All three monoclonal antibodies were confirmed to react with cOmpT in Western blot,without cross reaction with other tested bacteria.To preliminary identify the antigenic epitope recognized by those monoclonal antibodies,the compT gene without signal peptide was used as a template to design PCR primers according to the sequences of amino acids corresponding to different extracellular rings.After dual enzyme digestion,the resulting PCR products were cloned into the prokaryotic expression vector pET-28a.And then,the correct positive clones were induced for expression.The truncated expression proteins cOmpT A,cOmpT B,cOmpT C,cOmpT D,cOmpT E were detected by SDS-PAGE,and identified with monoclonal antibodies by Western blot analysis.The results revealed that the epitopes of 1G8 and 2C3 were identified on cOmpT B,and the epitope of 2G3 was initially located on cOmpT D.In order to narrow the antigenic epitope,a series of truncated cOmpT B and cOmpT D peptides were expressed.The results revealed that the antigenic epitope required for reactivity with the 1G8 was 83DQDWMDS89,2C3 was 90SNPGTW95 and 2G3 was 197TFKYSGW203.In this study,three monoclonal antibodies against cOmpT were successfully developed and the antigenic epitopes recognized by the antibodies were identified.The purpose of this study is to establish the material basis for the functional study of cOmpT.It could provide a basis for the development of APEC epitope vaccines. |
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