Development of a Scrub Typhus Diagnostic Platform Incorporating Cell-Surface Display Technology

Figures

• 

  Figure 1. Cloning strategies for surface display of recombinant TSA56 and ScaC-PD using a baculovirus-insect cell system. (A) Structure and strategy for displaying TSA56 protein on Sf21 cells. (A-a) TSA56 protein has a signal peptide from residues 1 to 22 and two TM domains (residues 67 to 87 and 472 to 492, respectively). (A-b) The signal peptide was replaced by an HM signal peptide and a His-tag, but the rest of the protein was preserved. (B) Structure and strategy for displaying ScaC-PD on Sf21 cells. (B-a) ScaC contains a signal peptide (SP), a passenger domain (PD), and an autotransporter (AT) domain. (B-b) The signal peptide (SP) was replaced by an HM signal peptide (HM) and a His-tag (His), and the TM-truncated passenger domain was fused with the TM and cytoplasmic-tailed domain (CTD) of GP64 protein (6MC) from baculovirus.

• 

  Figure 2. Schematic illustrations of transfer vectors used to construct recombinant baculoviruses. (A) The empty vector pAB-pEG-hhp10-HM-6H-6MC prior to TSA56 or ScaC-PD gene insertion. TSA56 from the Gilliam, Karp, or Kato strains, or ScaC-PD from Gilliam, were inserted into the vector, generating pAB-pEG-hhp10-HM6H-Gilliam (B), pAB-pEG-hhp10-HM6H-Karp (C), pAB-pEG-hhp10-HM6H-Kato (D), and pAB-pEG-hhp10-HM6H-ScaC-PD-6MC (E), respectively. EGFP, EGFP reporter gene; pag, pag promoter; hr1, homologous region; hsp70, heat shock protein 70 promoter; p10, p10 promoter; HM, honeybee melittin signal peptide; 6H, hexameric histidine tag; 6MC, transmembrane domain and cytoplasmic-tailed domain of baculovirus GP64 protein.

• 

  Figure 3. Validation of recombinant protein expression by Western blot analysis. Sf21 cells were infected with recombinant baculoviruses expressing TSA56 or ScaC-PD for 2 days. Expression of target proteins was assessed by Western blotting using anti-His antibody. GAPDH expression was determined as a loading control using anti-GAPDH antibody. Red arrow: TSA56 protein of the Gilliam and Karp strains. Yellow arrow: TSA56 protein of the Kato strain. Blue arrow: ScaC-PD. Wild-type: cells infected with wild-type baculovirus lacking foreign gene expression. Mock: cells not subjected to virus infection.

• 

  Figure 4. Immunofluorescence assay to confirm surface display of target proteins by Sf21 cells. Sf21 cells infected with recombinant baculoviruses carrying TSA56 or ScaC-PD gene constructs emitted green fluorescence arising from co-expression of the EGFP reporter gene. Cell surface display of recombinant TSA56 or ScaC-PD by the Sf21 cells was examined by immunostaining using primary anti-His antibody and secondary Alexa Fluor 555 antibody (red fluorescence). DAPI: nuclear counterstain (blue fluorescence). Wild-type: cells infected with wild-type baculovirus. Mock: cells not subjected to virus infection. Scale bar: 50 μm.

• 

  Figure 5. Validation of our cell-based ELISA using anti-His and anti-TSA56 monoclonal antibodies. (A) Schematic illustration of our cell-based ELISA. Sf21 cells infected by baculoviruses expressing TSA56 or ScaC-PD gene constructs were seeded into micro-well plates and treated with primary antibodies (Primary Ab) before adding secondary antibodies (Secondary Ab) that are conjugated with horseradish peroxidase (HRP). Cell-based ELISA treated with anti-His antibody (B) or commercial anti-TSA56 monoclonal antibodies YH560001, YH560010, or YH560012 (C). Wild-type: cells infected with wild-type baculovirus. Sf21: Sf21 cells not subjected to virus infection. ELISA reads are expressed as mean ± standard deviation (SD) of three replicates. Statistical significance was determined by Student’s t-test. *significant difference (p<0.05) versus the value for wild-type. †significant difference (p < 0.05) versus non-infected Sf21 cells.

• 

  Figure 6. Cell-based ELISA detection of ST in serum samples obtained from field-caught or non-ST rats. Sixty-nine rat sera confirmed as having been infected with O. tsutsugamushi by IFA (Positive) and thirteen negative control rat sera (Negative) were subjected to cell-based ELISA using cells displaying Gilliam TSA56 (A), Karp TSA56 (B), Kato TSA56 (C), and ScaC-PD (D) antigens, and cells infected with wild-type baculovirus (E). Individual data points are shown and the solid line represents the mean value. Dotted line: cutoff value of 0.056 determined as the mean value of negative rat serum reactivities against each of the antigens plus two standard deviations. P-values determined by Welch’s t-test are displayed above the plots.