A Preliminary Immunogenicity Study Of Serotype 1 Riemerella Anatipestifer OmpA/MotB Protein

Duck infectious serositis caused by Riemerella anatipestifer (RA) infection is one of the most serious infectious diseases in the duck industry, serotype 1 RA is the most widely popular. To explore the immunogenicity of OmpA/MotB protein, in this paper, the RA serotype 1 OmpA/MotB gene was bioinformatics analyzed, prokaryotic expressed and purified, ELISA detection method based on OmpA/MotB protein was established, immune protection effect was researched, obtained the following results:1.0mpA/MotB gene nucleic acid sequence and protein bioinformatics analysisVia bioinformatics software analysis and prediction, OmpA/MotB gene fragment length 1467 bp, whole is an open reading frame, no continuous 2 and over rare codon string. OmpA/MotB protein encoding 488 amino acids, it has OMP_b-brl subtribe, OmpA_C-like subtribe, OmpA three conservative district. No.1-21 amino acid residues as the signal peptide; No transmembrane region, is advantageous to the prokaryotic expression; Hydrophilic, soluble protein; Antigen epitope focused on no.127-470 amino acids (379bp-1410 bp); Subcellular localization in the cell’s outer membrane; Functional analysis could be a Receptor and Growth factors, can be involved in Stress response.2.0mpA/MotB protein prokaryotic expression, purification and western blotAccording to the OmpA/MotB gene sequence, designed two pairs of primers P1P2 and P3P4, extracted Riemerella anatipestifer serum type 1 genome, got gene fragments signal peptide removed and main antigen epitope area of OmpA/MotB by PCR amplification, respectively. Ligated into vector PMD19-T, after identified as being right, then cloned into expression vector PGEX-4T-1 and pET-32(a)+, respectively. Constructed the recombinant expression plasmid Successfully. Get the best expression condition of OmpA/MotB signal peptide removed protein was acquired as follows:0.4 mmol/L IPTG inducing for 12 h at 30 centigrade degree, its molecular weight was 76 kD, can be purified by sds-page rubber cutting way. The best expression condition of OmpA/MotB main antigen epitope protein was acquired as follows:1.2 mmol/L IPTG inducing for 12 h at 37 centigrade degree. The molecular weight was 56 kD, through the Ni2+-NTA agarose gel affinity chromatography purification. It confirmed that the two expressed proteins can be identified by Rabbit anti RA serotype 1 antibody through SDS-PAGE and immunoblotting.3.Development of indirect ELISA methods based on OmpA/MotB signal peptide removed and OmpA/MotB main antigen epitope areaThe best detection condition of OmpA/MotB signal peptide removed protein antibody level was coating with 3.61 μg/mL of OmpA/MotB protein overnight at 4 degree, and blocking 1 h at 37 degree with 5% dried skimmed milk, then incubate 2 h at 37 degree with 1:320 diluent of serum, following with 1:800 diluent of goat-anti-duck IgG antibody at 37 degree celsius for 0.5h and coloration for 10 min avoid light usingTMB. The positive threshold value was determined 0.15 with 40 samples of negative serum.Meanwhile, the best conditions based on OmpA/MotB main antigen epitope protein were coating with 2.9 p.g/mL for 3h at 37 centigrade degree,overnight at 4 centigrade degree, and 1% dried skimmed milk 2h at 37 centigrade degree for blocking,incubate with 1:160 diluent serum for 0.5h at 37 centigrade degree and then 1:1600 dilute IgG goat-anti-duck antibody for 0.5h at 37 centigrade degree,coloration for 25 min. And the positive threoshold was 0.12 with 40 samples of negative serum.Those two detection methods were highly specific, repeatable, sensitive. 4.Immunogenicity determination of OmpA/MotB protein for ducklingsPurified OmpA/MotB signal peptide removed protein and OmpA/MotB main antigen epitope protein were equally mixed with freund’s complete adjuvant respectively, then immunize 2 days cherry valley ducklings respectively. At the same time the commercialized vaccine group and control group were seted; commercialized vaccine group 1 feather doses and control group 0.5 mL saline injection per duck. Immune group were collected blood samples in the specified days, so as to determine OmpA/MotB signal peptide removed protein, OmpA/MotB main protein antigen epitope area, serotype 1 RA all bacteria ultrasound pyrolysis protein ELISA antibody levels, and IL-4, IFN-gamma, CD4+ and CD8+ levels were also detected. Immunization groups were challenged with serotype 1 RA in 16-day-old, the ducklings survived were executed on 23 days of age. Dissect ducklings of dead within one week and ducklings survived, meanwhile taked the heart, liver, brain tissue for bacteria isolation. The results show that OmpA/MotB signal peptide removed protein, OmpA/MotB main protein antigen epitope area have certain immune protection effect attacked by serotype 1 RA, can stimulate the ducklings to produce high levels of antibodies and a certain levels of cytokines. It suggests that serotype 1 RA OmpA/MotB protein has good immunogenicity, can stimulate the body to produce humoral immunity and cellular immunity simultaneously.