Establishment Of Indirect ELISA Method For Salmonella Antibody Detection From Ducks Based On PagN Protein

China is the country with the largest duck farming scale in the world.In recent years,relevant reports showed that there was a marked increase in the prevalence of Salmonella in ducks,which has brought serious economic losses to the duck industry in China.Salmonella can be transmitted horizontally and vertically among poultry such as chickens and ducks.Moreover,adult animals are often latently infected,which facilitates the spread of Salmonella and poses great challenges for control.In this study,a highly conserved and specific protein PagN in Salmonella was screened by bioinformatics analysis,and it was expressed in vitro with high purity degree.Finally,an indirect ELISA method was established based on recombinant PagN protein for the detection of Salmonella antibody from ducks.The method can accurately assess Salmonella contamination in duck flocks,it is necessary to provide a reliable basis for the prevention,control and purification of Salmonella,and contribute significantly to promote healthy development of the duck industry.1.Screening of target proteinThe target protein PagN was successfully screened by bioinformatics and genetic evolution analysis.Through BLASTN comparison,we found that the homology of PagN gene in Salmonella reached more than 98%,and the homology with other non-Salmonella was less than 50%.DNA Star software predicted that the PagN protein had a relatively large proportion of β-turn and random coil,and contained good hydrophilicity,flexibility,immunogenicity and surface possibility.Epitope prediction showed that four antigenic epitopes with strong antigenicity nearly distributed in the extracellular loop region of the protein,which could stimulate the body to produce specific antibodies for serological detection.2.Recombinant expression of PagN proteinIn this study,the complete DNA sequence of PagN gene was obtained by PCR,and cloned into p ET-32a（+）to construct successfully the recombinant prokaryotic expression plasmid p ET-32a（+）-PagN.The recombinant plasmids confirmed by enzyme digestion and target sequenc was transformed into E.coil BL21,and then induced by IPTG（1 m M）for 6 h at 37°C with shaking 220 rpm.SDS-PAGE analysis showed a single band corresponding to the molecular weight of 43 k Da.Our analysis indicated that PagN protein was highly expressed in the form of inclusion bodies in E.coli.After solubilization of inclusion bodies in urea,we purified recombinant PagN protein using affinity chromatography,and then removal of urea by gradient dialysis facilitated the renaturation of protein.Protein solutions were prepared in the adsorption buffer with final concentrations of 1.052 mg/m L.Western blotting analysis revealed that recombinant PagN protein showed good reactogenicity,and has been verified as a detecting antigens using for indirect ELISA.3.Establishment of the indirect ELISA detection method based on recombinant PagN proteinWe initially established the indirect ELISA assay for detecting Salmonella antibodies based on recombinant PagN protein,and determined the optimal reaction conditions: the concentration of coating antigen was 1 μg/m L,coating for 2 h at 37°C;5% skimmed milk powder blocked for 2 h at 37°C;the best serum dilution was 1:50,the incubation condition of serum was at 37°C for 30 min;the enzyme-labeled secondary antibody at a working dilution of 1:4,000 was incubated 90 min at 37°C,and the catalytic reaction time of TMB was 20 min.In the present study,we obtained a fixed cut-off of 0.268 for ELISA performance by detecting 38 Salmonella negative serum samples.The method has high specificity sensitivity when treated with S.Enteritidis,S.Typhimurium and S.Kottbus positive duck sera.At the same time,EILSA method had no cross-reaction with antibodies against E.coli,Staphylococcus aureus and Streptococcus.The method had a minimum detection limit of 1:1,600 for the positive sera against Salmonella.The coefficients of variation in repeated experiments were all below 10%.ELISA method and Dot-blot test were performed on 100 positive sera collected from duck farms,and the total coincidence rate was89%（89/100）.613 duck serum samples from 3 different farms were tested using established method,and the overall positive rate was 44.86%.The indirect ELISA antibody detection method based on recombinant PagN protein was established in this study,demonstrating a high sensitivity,specificity and be suitable for the detection of Salmonella with various serotypes,which provided a new research direction for clinical detection of duck-derived Salmonella and reliable technical support for the development of antibody detection kit.