Bacterial strains and culture conditions
RA strain ATCC 11845 were purchased from Shanghai yiyan biological technology Co. Ltd (ShangHai, China). Serotype 1 RA strain CH-1 (RA-CH1) was isolated from sick ducks in HaiKou, China. RA strains were grown in tryptic soy broth medium (TSB) and at 37°C. E. coli strain DH5α and BL21 (DE3) strains were purchased from TransGen Biotech, Inc. (Beijing, China). E. coli strains were grown on Luria-Bertani (LB) agar.
Animals
30 One-day-old Cherry Valley ducklings obtained from the Hainan Tianyuan Industrial Co., Ltd.(Haikou, China). The ducks were housed in cages, and provided water and food adlibitum during the study. The animal experiments were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Institutional Animal Care and Use Committee guidelines set by HanNan University.
Signal peptide prediction
Signal peptide cleavage site was predicted by SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP/).
Homology analysis and phylogenetic tree construction
The RA Porin protein (Uniprot NO. E4TDA8) was blasted by UniProtKB database with parameters including E-Threshold 10, Matrix BLOSUM-62 and no filtering in Uniprot (https://www.uniprot.org/blast/). The number of returned alignments was 50. The blasted data was aligned with ClustalW and phylogeny tree was constructed by MEGAX.
Transmembrane structure prediction
Mature peptide sequence of RA Porin protein was pasted into PRED-TMBB web server (http://biophysics.biol.uoa.gr/PRED-TMBB/input.jsp)(Bagos, Liakopoulos, Spyropoulos, & Hamodrakas, 2004), which is for predicting the topology of beta-barrel outer membrane proteins. Viterbi (VI), N-best (NB) and Posterior Decoding (DY) method were applied and the common part of the three analyses was as prediction results.
Tertiary structure prediction
The tertiary structure of the Porin protein sequence from which the signal peptide was removed was predicted by the swissmodel online server (https://swissmodel.expasy.org/).
Antigenic prediction
The B cell epitopes were predicted by ElliPro web server (http://tools.iedb.org/ellipro/) based based upon structural protrusion. The predicted results were screened to retain only epitopes outside the membrane.
Expression and purification of poring protein of RA
The RA genome is extracted by the genome extraction kit (TIANGEN Biotech(Beijing)Co.,Ltd.,BeiJing, China), and the porin gene is amplified by primer pairs porin F: 5’-GGATCCGTACCAGATACTATTATCGTTC-3’ and porin R: 5’-GCGGCCGCAATTCCTATTTCTACTTGAAATCT-3’, with Restriction site BamH 1and Not1. The gene was then ligated into the pET 28a vector, and the recombined vectors were transformed into BL21 competent cells. These successfully transformed BL21 E. coli cells were induced to express the target protein by auto-inducing medium(Studier, 2014). The expressed product was subjected to cleavage, separation, denaturation, renaturation, dialysis, and finally purification through a nickel column based on the previous protocol(Saleem, Moore, & Derrick, 2012). All proteins were determined by 12 % sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS–PAGE). The protein was then lyophilized with the ALPHA 1-2 LD plus freeze dryer (Christ, Germany) and kept in −20 °C.
Immunization protocols
The Cherry Valley ducks were randomly divided into 3 groups of 10 animals each injected with PBS (PBS group), PBS + adjuvant (PBS + adjuvant group), PBS + adjuvant + Porin protein (Porin group). The ducks were first immunized at 7 days of age. The Porin protein was mixed with Freund's complete adjuvant in a 1:1 oily form and injected subcutaneously. For the second immunization 7 day post the first immunization, Porin protein was mixed with Freund's incomplete adjuvant by 1:1 to form an oil emulsion. Both immunization doses were 0.2 mg/duck. The days of blood collection was 1, 7, 14, 21 and 28 days post the first immunization.
Indirect enzyme-linked immunosorbent assay (iELISA) for Porin protein and RA cells detection
96-well plates were coated at a concentration of 2 μg/ml protein antigen with 100 μl/well, overnight at 4 ° C, and washed 4 times with 100 μl PBST wash for 5 min each. The plates were added with 200 μl/well blocking solution and incubaed at 37 ° C for 2 hours. Then the plates were washed 4 times with each time 5 min. The first well was diluted 1:100, diluted 1:3 in a gradient, 100 μl/well. The plates were then incubated at 37 °C for 1.5 h, and washed 4 times for 5 min each time. Horseradish-labeled mouse anti-duck IgG (baiaolaibo, Beijing) was diluted 1:5000, 100 μl/well, incubated at 37 ° C for 20 to 30 min, and washed 4 times for 5 min each time. The plates were added with 1×TMB at 100 ul/well, and then incubated for 15-30 min at 37 °C. Finally, stop solution (2M H2SO4) was added into the plates at 50 μl/well.
The OD value of each well was measured at a single wavelength of 450 nm, and the ratio (Positive/Ngetive, P/N) of the OD value to the negative control well was greater than 2.1 as a critical point judged to be positive or negtive.
96 well plates were added with 150 μl 0.1 M NaHCO 3 plus 2.5 % glutaraldehyde, incubated for 1 h at 37 °C, and washed four times with sterile water. The plates were then coated with 107 CFU/100 μl RA cells per well and incubated at 37 °C until dry. Subsequent steps from antigen blocking were carried out in accordance with the above iELISA procedure.
Serum bactericidal assay (SBA)
The serum 7 days after the second immunization was used for SBA. A single colony of the purely cultured strain was picked, inoculated into TSB medium, and cultured to the early stage of growth (OD 600 = 1.5 or so). After washing with PBS, it was diluted to 108 cfu/ml.Duck serum was diluted to a range of concentrations of 100%, 50%, 25%, 12.5%, 5% with PBS. Inactivated 100% duck serum and PBS were used as controls. 10 uL of the prepared bacterial solution was mixed with 190 uL of different concentrations of duck serum and incubated at 37 ° C for 30 min. The number of bacteria survived was counted by plate counting.
Opsonophagocytosis assay
The serum 7 days after the second immunization was used for Opsonophagocytosis assay. RA single colonies were picked, inoculated into TSB liquid medium, and cultured to the early stage of growth (OD 600 = 1.5 or so). After washing with PBS, it was diluted to 5 x 104 CFU/ml. 20 μl of the test serum was added to the 96-well cell plate and incubate for 20 min at 37 °C. 10 μl of duck complement and 40 μl of duck blood was add into the plates and incubate for 1 hour at 37 °C with shaking. The number of bacteria survived was counted by plate counting.
Challenge assay
30 one-day-old cherry valley ducks were randomly divided into three groups. Porin protein with adjuvant, PBS with adjuvant and PBS were separately immunized according to the previous method. After 14 days of secondary immunization, 10 times and a half lethal dose (LD50) RA CH1 was intraperitoneally injected for 1 week, and survival rate after challenge was recorded.
Statistical analysis
The SigmaPlot software (version 12) and graphpad prism 8.0.2 were used for all statistical analyses. One-way repeated analysis of variance (ANOVA) and the Mann–Whitney rank test were used to evaluate differences between groups. Differences were considered significant at p < 0.05.