Bacterial strains, plasmids and growth condition
Escherichia coli O86:B7 (ATCC 12701) was obtained from American Type Culture Collection (Rockville, MD). Commercially available IgM monoclonal anti-B antibody (obtained from clone HEB-29) was purchased from Merck Millipore (Billerica, USA). All strains and plasmids used in this study were listed in Additional file 1: Table S1. All strains were grown in Luria–Bertani broth (LB) at 37 °C. E. coli DH5α and O86 were used for plasmids cloning and glycoprotein expression experiments, respectively. Ampicillin (100 μg/mL), 50 μg/mL kanamycin and 34 μg/mL chloramphenicol were added to the media for selection as needed. Plasmids pKD4, pKD46 and pCP20 were used for the deletion of the gene coding for the O-polysaccharide ligase WaaL of E. coli O86. Plasmid pACT3 and pBAD24 were used for the expression of PglB and MBP protein, respectively.
Knockout of waaL gene of E. coli O86
The waaL gene of E. coli O86 was knocked out to obtain O86 ΔwaaL: FRT using λ-Red recombination system. Briefly, using plasmid pKD4 as template, the kanamycin-resistant gene flanked by homologues of waaL gene was amplified by PCR with knockout primers. When induced by L-arabinose, plasmid pKD46 could express three recombinant proteins (Exo, Beta, Gam) of λ-prophage, which assisted the replacement of waaL gene with kanamycin-resistant gene. Subsequently, the kanamycin-resistant gene was eliminated by FLP-promoted recombination system using plasmid pCP20 and the E. coli O86 ΔwaaL was obtained successfully. The knockout primers (k-waaL-F, k-waaL-R) and test primers (t-waaL-F, t-waaL-R) used in the knockout experiments were listed in Additional file 1: Table S1. The extraction of LPS was carried out according to the instruction of LPS extraction kit (iNtRON Biotechnology, KOREA). The silver staining experiment was performed as reported previously [19].
Construction of recombinant plasmids
In order to ensure the successful glycosylation of MBP by PglB, the consensus sequence D-Q-N-A-T was repeated four times and inserted at the C terminal of MBP. Overlap PCR was used to amplify the malE
mut gene with primers malE-F, malE-R1, malE-R2 and malE-R3 (Additional file 1: Table S1). Restriction sites for Sal I and Hind III at their 5′ ends of primers were used for the insertion of the modified gene into the vector pBAD24, and thus the plasmid pBAD24-malE
mut was obtained with a 6× His tag (i.e. N-HHHHHH-C) between Sma I and Sal I of pBAD24 (Induced by L-arabinose). Likewise, the pglB gene from C. jejuni NCTC 11168 was inserted between Sma I and Sal I of plasmid pACT3 (Induced by IPTG) and the plasmid pACT3-PglB was obtained.
Glycoprotein expression and purification
The recombinant plasmids pBAD24-malE
mut and pACT3-PglB were co-transformed into E. coli O86 ΔwaaL to obtain an engineering strain with the ability to produce MBPmut-OPS bioconjugates. Plasmid containing MBP
mut
gene was transformed into E. coli O86 ΔwaaL to produce unglycosylated MBPmut as a control. E. coli O86 ΔwaaL transferred with pBAD24-malE
mut and pACT3-PglB was grown in 50 mL LB broth at 37 °C for 16 h, with shaking. Cultures were then inoculated 1/100 into 1 L TB broth and further grown at 37 °C with shaking until OD600 reached 0.6. Subsequently, 0.1 % (w/v) L-arabinose and 50 μM IPTG were added to induce the expression of MBP and PglB, respectively. After further incubation at 28 °C for 6 h, 0.1 % (w/v) L-arabinose was added again for continuous induction of MBP.
After that, cells were pelleted by centrifugation at 10,000 rpm for 15 min at 4 °C, and then resuspended in lysis buffer (50 mM PBS, 200 mM NaCl, 5 % glycerin, pH 7.4). The supernanant of cells after ultrasonic lysates was purified using pre-equilibrated Ni-nitrilotriacetic acid (NTA) columns under native conditions. Washing buffer (50 mM PBS, 200 mM NaCl, 5 % glycerin, 50 mM imidazole, and pH 7.4) and elution buffer (50 mM PBS, 200 mM NaCl, 5 % glycerin, 250 mM imidazole, and pH 7.4) were sequentially used. Fraction containing the purified glycoconjugate was collected and then desalted using centrifugal filter (Amicon® Ultra-15, Milipore) against PBS (PH 7.4). The concentration of the proteins was measured with Bradford method.
Detection of purified glycoprotein
Western blotting was used to detect MBP and MBPmut-OPS expression. Samples were separated on 8 % SDS-denatured polyacrylamide gel and were then transferred onto nitrocellulose membrane. Membranes were blocked in 3 % BSA solution for 1 h at room temperature, and then were incubated with anti-hexahistine (anti-His) monoclonal antibody and anti-MBP monoclonal antibody (Beyotime Biotechnology, China), as well as anti-O86 O-antigen polyclonal antibody (Tianjin Biochip Corporation, China), respectively overnight at 4 °C. The secondary antibodies with a horseradish peroxidase (HRP) (Abcam, UK) were used subsequently. The image acquisition was finished by Flour ChemQ (Proteinsimple, US). MALDI-TOF result was analyzed by the MALDI-TOF mass spectrometer (AXIMA Confidence, SHIMAZU, Japan) with sinapic acid as the matrix (50 % ACN, 50 % H2O, 0.1 % TFA).
Binding ability measurement of glycoprotein and anti-B antibody
Polystyrene microtiter plates were coated by the purified proteins MBP/MBPmut-OPS from E. coli O86 at different concentration overnight at 4 °C. The plates were blocked with 2 % BSA in PBS buffer for 2 h at room temperature. After being washed three times with PBST (PBS, 0.05 % Tween-20), the plates were incubated with anti-B antibody diluted to 1:20 for 2 h, or with anti-A antibody (1:20) as control. After washing, the secondary antibody goat anti-mouse IgM conjugated to HRP (1:20,000) (Abcam, UK) was added and maintained for 1 h. Finally, the TMB substrate was used to develop the signal and 1 M HCl was used to terminate the reaction, and the OD was measured at 450 nm on Bio-Rad680 microplate reader (Hercules, California, USA).
Detection of the B antibody titer and coagulation parameters in the plasma
All blood samples, from 36 healthy people, were collected with citrate anticoagulation tubes, mixing, and were centrifuged at 1000g for 10 min to separate plasma. The plasma was divided two portions, one for the detection of B antibody titers, the other for coagulation analysis.
The B antibody titers in the plasma were measured with the polybrene test according to the instruction (Baso Biological Technology Corporation, Zhuhai, China). Briefly, twofold serial dilutions of plasma sample from 1:2 were made with normal saline for each tube. The same volume of 2 % type Bred blood cells were added to each tube and mixed thoroughly. Low ionic medium, polybrene reagent and resuspending were added subsequently and operated based on the instruction, and the smallest dilution which could still agglutinate erythrocyte was determined as the endpoint, and its reciprocal was considered as the titer of the sample plasma.
In order to detect the effects of proteins MBPmut-OPS on blood clotting function, coagulation parameters of the samples treated/pre-treated with MBPmut-OPS were measured with fully automatic blood coagulation analyzer ACL7000 (BECKMAN, USA).
Adsorption of blood group B antibody in the plasma
Aliquots of plasma samples of 800 μL were mixed with final concentration of 0, 80, 160 and 320 μg/mL MBPmut-OPS, respectively. After incubation at room temperature for 1 h, the B antibody titer and clotting parameters in the plasma were detected as above methods.
Statistical analysis
The statistical analyses and figures were generated by GRAPHPAD PRISM software version 5.0. Data were shown as mean ± standard deviation (SD). The difference between two groups was compared by t test. For multiple comparisons, One-way ANOVA was used. A probability (P) value ≤ 0.05 was considered statistically significant.
