Study On The Adhesion Function And B Cell Epitopes Of The GapA Protein From Nocardia Seriolae

In recent years,fish diseases caused by Nocardia seriolae（N.seriolae）,which are characterised by skin ulcers and nodules in internal organs,have seriously endangered the healthy development of the aquaculture industry,and it is urgent to develop safe and efficient vaccines for the prevention and control of the disease.Bacterial adhesion is a key initial step in pathogen infection,and the vaccine aimed to adhesin play an antibacterial role in the initial stage of infection.In our previous study,a pathogenic N.seriolae was isolated and identified from diseased Micropterus salmoides.It is found that the isolate carried a highly conserved gap A gene encoding Glyceraldehyde-3-phosphate dehydrogenase（GapA）through genome sequencing and analysis,and it is predicted that GapA might posses adhesion function,but it still needs to be verified.Therefore,GapA protein was taken as the research object and CIK cells was used as the adhesion model to explore the adhesion function of the protein,identify its B cell epitope and determine its epitope of adhesion function in the study.The purpose of this research is to lay the foundation for the later development of multiepitope-based adhesin vaccine.The main research contents and results are as follows:1.Recombinant expression of N.seriolae GapA proteinIn this study,gap A gene was PCR amplified from genomic DNA of N.seriolae and cloned into plasmid p MD19T to constructe p MD19T-gap A,and then constructed p ET32a（+）-gap A by enzyme digestion and linked.The correct recombinant strain BL21/p ET32a（+）-gap A was induced by IPTG.The expression product was denatured,renaturation,purification and concentration,respectively,and r GapA protein with purity of 93%and concentration of 1.14 mg/m L was obtained.2.Preparation of rabbit anti-r GapA antibody and rabbit anti-N.seriolae antibodyIn this study,the recombinant GapA protein,oil adjuvant and immunopotentiator were mixed and emulsified as immunogen to immunize New Zealand white rabbits to prepare rabbit anti-r GapA antibody.The results showed that the titer of rabbit anti-r GapA antibody,which could specifically recognize GapA protein,was 1:32 detected by immunodiffusion test.Similarly,rabbit anti-N.seriolae antibody with agglutination titer of 1:1 024 was prepared as above method.These two antibodies can be used for subsequent detection.3.Studies on adhesion of the GapA protein from N.seriolaeIn this study,the adhesion of r GapA protein to CIK cells was detected by immunofluorescence,Western blot and bacterial adhesion inhibition test,respectively.The results showed that green fluorescence appeared on the cytomembrane of CIK cells pre-incubated with r GapA protein,but not on the cytomembrane of CIK cells pre-incubated with the mixture of r GapA protein with r GapA antibody.The cytomembrane protein extracted from cells post-treatment with r GapA protein could bind to the anti-r GapA antibody,showing a reaction band with a size of about 53.6 k Da.The mean number of adhesion bacteria around CIK cells in bacterial adhesion control group,antibacterial antibody treatment group and anti-r GapA antibody treatment group were21.60±5.70,0.00 and 5.60±0.49,respectively,the adhesion inhibition rate of r GapA antibody was 74.10%.These results indicated that r GapA protein could adhere to CIK cells.4.Identification of B cell epitopes and adhesion peptides of the GapA proteinIn this study,DNAStar Protean and Pymol software were used to predict the dominant B cell epitopes of GapA protein,which were then verified by peptide ELISA and Western blot.The result showed that these predicted epitopes,Ep_58-69（RLPQDVSLDGDY）,Ep_139-150（GVNDDKYDGSQN）,Ep_186-197（YTQDQNLQDAPH）and Ep_318-329（DNEWGYSNRLAD）,were identified as the B cell epitopes of GapA protein.Then,the FITC-labelled epitope peptides and the irrelevant control peptide were artificially synthesized,and adhesion of these labelled peptides to CIK cells was detected by flow cytometry.The results showed that the percentage of FITC-labelled positive cells post incubation with each labeled peptide was 1.09±0.07%(Ep_58-69),97.40±0.57%(Ep_139-150),98.10±0.49%(Ep_186-197),86.30±1.71%(Ep_318-329)and 1.12±0.07%（irrelevant peptide）,the corresponding mean fluorescence intensity was 2.73±0.35,57.08±4.12,38.82±1.62,39.49±0.82 and 2.35±0.20,respectively,suggesting that Ep_139-150,Ep_186-197and Ep_318-329could adhere to CIK cells,which were epitope peptides with adhesion of GapA protein.In conclusion,on the basis of successful preparation of rabbit anti-r GapA antibody,it was confirmed that GapA protein is an adhesin of N.seriolae in the study,there were four B cell epitopes of GapA protein were identified for the first time,and these epitopes,such as Ep_139-150,Ep_186-197and Ep_318-329,were determined as epitope peptides with adhesion.Our findings lay a good foundation for the later development of multiepitope-based adhesion vaccine against N.seriolae.