cord blood sampling
Cord blood samples were obtained in 10-ml heparin tubes (BD Vacutainer), Becton, Dickinson & Company, Franklin Lakes, NJ, USA). All relevant guidelines and regulations were followed, and all experimental protocols involving human samples were approved by the Kangwon National University Hospital Ethical Committee (IRB No. KNUH-B-2021-06-034). Written informed consent was obtained from all subjects.
Recombinant protein expression and purification
All recombinant proteins were produced using the mammalian human embryonic kidney 293E (HEK293E) cell expression system as described elsewhere (28). For plasmid preparation, pTT3 and pTT5 containing the appropriate exogenous signal peptide with the desired C-terminal tag were generated. A group of reticulocyte receptor proteins and parasite ligand proteins were included with Fc- and His-tagged, respectively. The full-length ectodomain was selected for expression, except P. vivax reticulocyte binding protein 2B (PVRBP2B, residues 169 to 813), P. vivax GPI-anchored micronemal antigen (PVGamma, residues 408 to 589), PVDBP (region II, residues 194 to 521), P. vivax merozoite surface protein 1 paralog-19 (PVMSP1p-19, residues 1751 to 1834), and DARC (CD234, residues 1 to 63). DNA (cDNA) complementary to the expected sequences was codon-optimized for expression in mammalian cells using GeneArt (https://www.thermofisher.com/order/geneartgenes/projectmgmt) and then processed by Twist Bioscience (South San Francisco ) was chemically synthesized by. Reason). HEK cells were maintained in suspension in Gibco Freestyle™ F17 expression medium (Life Technologies Corp., Grand Island, NY, USA) at 37 °C under 70% humidity and 8% CO.2 In an orbital vibration incubator at 120 revolutions per minute. Twenty-four hours before transfection, fresh F17 medium was used to seed cells to a final density of 6 to 8 × 105 cells/ml. For each transfection, the expression plasmid was mixed with Polyethyleneimine Max transfection reagent (Polysciences, Warrington, PA, USA) before transfection into HEK cells. To produce a secreted form of the biotinylated protein, D-biotin (Sigma-Aldrich, St. Louis, MO, USA) and a plasmid encoding Escherichia coli Biotin ligase (BirA) was added during transfection. After 5 days of incubation after transfection, the culture supernatant containing proteins was collected for protein purification. His-tagged proteins were purified using Ni-NTA agarose (QIAGEN, Hilden, Germany) and a Poly-Prep. Chromatography column (Bio-Rad, Hercules, CA, USA). hightrap Proteinase G HP (Sigma-Aldrich) was used for purification of Fc-fusion proteins. The purified proteins were subjected to buffer exchange with HEPES-buffered saline (HBS) and concentrated using a 30-kDa Amicon. Ultra-15 centrifugal filter (Sigma-Aldrich).
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.
His- and Fc-tagged recombinant proteins were separated by 13% SDS-PAGE. When required, a reducing agent was added to the sample to induce reducing conditions. Gels were stained with Coomassie brilliant blue (Sigma-Aldrich) for SDS-PAGE analysis, while others were used for protein adsorption onto polyvinylidene fluoride (PVDF) membranes (MilliporeSigma, Burlington, MA, USA), with After the membrane was blocked. 5% skim milk for 1 hour. Membranes containing His-tagged proteins were incubated with anti-Pena-His antibody (1:2000) (LI-COR). Biosciences, Lincoln, NE, USA) and then with a secondary IRDE Goat anti-mouse (1:10,000) antibody (LI-COR Biosciences), whereas for Fc-fusion protein (LI-COR) only goat anti-human antibody (1:5000) was used Biosciences). Data analysis was performed with the Odyssey Infrared Imaging System and Software (LI-COR) Biosciences).
Protein-protein interaction screening by enzyme-linked immunosorbent assay (ELISA)
Biotinylated proteins were normalized with mouse anti-rat CD4 monoclonal antibody (clone: OX68, Novus Biologicals™, Centennial, CO, USA) on a streptavidin-coated plate. After normalization, 100 μl of each biotinylated protein was incubated for 1 h at room temperature (RT) and then washed with phosphate-buffered saline with 0.1% Tween 20 (PBS-T) and PBS. Then, 1 μg of Fc-fusion protein in PBS-T was added, and the mixture was incubated for 1 h with shaking. After washing steps with PBS-T, the plate was allowed to react with goat anti-human Fc-specific alkaline phosphatase antibody (Sigma-Aldrich, 1:5000 in PBS-T) for 1 h at RT. . After washing with PBS-T, 100 μl of 1-Step™ p-nitrophenyl phosphate disodium salt (PNPP; Thermo Scientific, MA, USA) solution was added. The plate was incubated for 30 min at RT, and then the absorbance was measured at 450 nm.
Reticulocytes were enriched from umbilical cord blood using 19% Nicodenz solution (Axis-Shield, Oslo, Norway) in high-KCl buffer with gradient centrifugation as previously mentioned (29). Briefly, fresh umbilical cord blood was washed twice with incomplete Roswell Park Memorial Institute (RPMI) 1640 medium, and white blood cells were separated through a non-woven fabric (NWF filter (Zhixing Bio S&T Co., Ltd., Bengbu, China ) was removed by centrifugation, packed cells were re-suspended in high-KCl buffer (115 mM KCl, pH 7.4) and then incubated at 4 °C for 3 h with rotation. Each 5 ml mixture of red blood cell (RBC)-high-KCl buffer was overlaid on Nicodenz solution (19%, 3 ml) in a 15 ml tube followed by centrifugation at 3000×.Yes For 30 min without braking, reticulocytes enriched in the interface layer were collected and washed three times with incomplete RPMI 1640 medium. The purity of reticulocytes was evaluated using thin blood smears stained with new methylene blue with light microscopy and flow cytometric analysis using thiazole orange (TO) (Becton Dickinson, San Jose, CA, USA). A total of 100,000 events were obtained from each sample using fluorescence-activated cell sorting (FACS) Accuri™ C6 flow cytometer (Becton Dickinson, Mansfield, MA, USA).
Flow cytometric analysis of CD36 expression on erythrocytes
Samples for flow cytometric experiments were prepared as described previously with slight modifications (24). Enriched reticulocytes were washed three times with PBS containing 1% bovine serum albumin (BSA), and then 1 million RBCs were treated with 5 μg PE/cyanine 7 anti-human CD36 (BioLegend, San Diego, CA, USA) or similar. Was incubated with. Quantitation of PE/Cyanin7 mouse IgG2 α,κ isotype control antibody as a control (BioLegend) for 30 min at 4 °C in the dark. After washing three times with PBS-1% BSA, cells were incubated with TO for 30 min at RT. A total of 100,000 events per sample were acquired using a FACS Accuri™ C6 flow cytometer.
Flow cytometry-based erythrocyte binding assay
Reticulocyte binding assay was performed as described previously (30). In short, 1×106 Cells were incubated with serial concentrations of purified His-tagged recombinant PvMTRAP for 3 h at 25 °C. PvRBP2b-His and glutathione S-transferase (GST)-His proteins were used in parallel as positive and negative controls, respectively. After incubation, samples were washed twice with 200 μl of PBS-1% BSA and then incubated with 5 μL of APC anti-His tag monoclonal antibody (BioLegend, San Diego, CA, USA) in a 100 μl staining volume for 1 h. Incubated with μg. 4°C in darkness. Samples were washed three times with PBS-1% BSA and incubated with TO for 30 min at 25 °C. A total of 100,000 events per sample were counted using a FACS Accuri™ C6 flow cytometer. All flow cytometric data were analyzed by FlowJo software (TreeStar, Ashland, OR, USA).
octet bio-layer interferometry analysis
Conversation between their-tagged P. vivax Ligands and Fc-tagged reticulocyte receptors were evaluated using the protein G (ProG) biosensor in the Octet RED96 system (Fortebio Inc., Menlo Park, CA, USA). Fc-tagged reticulocyte receptors were immobilized on the ProG biosensor, and then a two-fold serial dilution of the parasite ligand in HBS was loaded into the biosensor to generate kinetic parameters. Data were analyzed with Octet Systems data analysis software ver. 7.0. (Sartorius, Gottingen, Germany).
Data were calculated using GraphPad Prism (GraphPad Software, San Diego, CA, USA) and Microsoft Excel 2016 (Microsoft, Redmond, WA, USA). For comparison, paired students Tea-tests were used, and a Why-value <0.05 was considered to indicate significance.
(TagstoTranslate)Parasitology(T)Entomology(T)Tropical medicine(T)Infectious diseases(T)Veterinary medicine/veterinary science(T)Virology