Screening for a single-chain variable-fragment antibody that can effectively neutralize the cytotoxicity of the Vibrio parahaemolyticus thermolabile hemolysin

Abstract

Vibrio parahaemolyticus is a halophilic bacterium that is widely distributed in water resources. The bacterium causes lethal food-borne diseases and poses a serious threat to human and animal health all over the world. The major pathogenic factor of V. parahaemolyticus is thermolabile hemolysin (TLH), encoded by the tlh gene, but its toxicity mechanisms are unknown. A high-affinity antibody that can neutralize TLH activity effectively is not available. In this study, we successfully expressed and purified the TLH antigen and discovered a high-affinity antibody to TLH, named scFv-LA3, by phage display screening. Cytotoxicity analysis showed that scFv-LA3 has strong neutralization effects on TLH-induced cell toxicity.

Fig 1

Expression of recombinant TLH in E. coli. SDS-PAGE analysis was performed with recombinant TLH expressed by plasmids pET32a(+) (A) and pET28a(+) (B). Lanes: M, protein molecular mass markers; 1, negative control (empty vector); 2, total cell lysate; 3, TLH purified using a Ni²⁺-nitrilotriacetic acid (Ni²⁺-NTA) column.

Fig 2

TLH induces cytotoxicity in cultured cells. (A) HeLa cells (a and b), Changliver cells (c and d), and RAW264.7 cells (e and f) were treated with or without 20 μg/ml TLH. Cell morphology was observed under a microscope and photographed after 24 h. Magnification, ×100. (B) Dose-response analysis of TLH-mediated cytotoxicity. HeLa, Changliver, and RAW264.7 cells were exposed to different concentrations of TLH for 24 h, and cell viability was determined using the MTT assay. Data are presented as means ± standard deviations (SD) for three separate experiments. (C) Time course of TLH-mediated cytotoxicity. HeLa, Changliver, and RAW264.7 cells were incubated with 20 μg/ml of TLH for the indicated times. Cell growth was determined using the MTT assay. (D) Serum titer assay. ELISA was used to determine the serum titer after four immunizations. The serum titer was evaluated by the S/N ratio (signal ≥1.0); the cutoff value for a positive clone was an S/N ratio of ≧2.

Fig 3

Construction of an anti-TLH scFv antibody library. (A) PCR analysis of V_H and V_L. Lane 1, V_H gene (∼340 bp); lane 2, V_L gene (∼325 bp); lane M, DL-2000 DNA marker. (B) Amplified fragment of the scFv gene. Lane M, DL-2000 DNA marker; lanes 1 and 2, amplified scFv gene (∼750 bp). (C) Selection of phage scFv antibody library by panning. In each round of panning, the number of input phage was kept constant, at 3.0 × 10¹⁰ CFU/ml, and phage that did not bind TLH was removed by washing. The number of phage was counted after each panning round. After three panning rounds, the number of eluted phage was kept constant at 3.0 × 10⁶ CFU/ml. (D) ELISA analysis of the binding activities of four different anti-TLH scFv antibody clones. The graph shows the relationship between OD₄₅₀ values and TLH concentrations.

Fig 4

Molecular characterization of scFv-LA3 clone. (A) Nucleotide and amino acid sequences of the scFv-LA3 fragment. Complementarity-determining regions are underlined. (B and C) IMGT collier de perle graphical 2-dimensional representations of the V_H (B) and V_L (C) regions of scFv-LA3. Asterisks indicate differences between scFv-LA3 and mouse germ line genes. Hydrophobic amino acids (those with positive hydropathy index values, i.e., I, V, L, F, C, M, and A) and tryptophan (W) are shown with gray circles. All proline (P) residues are also shown in gray circles. The CDR IMGT sequences are delimited by amino acids shown in squares (anchor positions), which belong to the neighboring framework region (FR-IMGT). Dotted circles correspond to missing positions according to the IMGT unique numbering. Residues at positions 23, 41, 89, 104, and 118 are conserved.

Fig 5

Characterization of scFv-LA3. (A) SDS-PAGE analysis of expressed scFv-LA3. Lane M, protein marker; lane 1, negative control (empty vector); lanes 2 and 3, expression product of scFv-LA3. (B) Western blot analysis of the binding activity of scFv-LA3 toward the TLH antigen. (Left) SDS-PAGE results for Ni²⁺-NTA-purified TLH. Lane M, protein molecular mass markers; lanes 1 and 2, purified TLH. (Right) Western blotting results. Lanes 3 and 4, TLH band at 47 kDa bound by scFv-LA3. The bound anti-TLH scFv-LA3 clone was detected using an anti-E-tag HRP-conjugated antibody. (C) scFv-LA3 specifically binds the TLH antigen. *, P < 0.05. BSA and associated antigen proteins (TDH, TLH, and YscF) of Vibrio parahaemolyticus were used to coat 96-well plates in triplicate (5 μg/ml; 100 μl/well). Secreted recombinant phage particles were added to the reaction wells and incubated for 2 h at 37°C. Specificity of the scFv-LA3 clone was determined using an anti-M13 HRP-conjugated antibody. (D) Western blotting. The total proteins isolated from tlh-positive and tlh-negative bacterial strains were transferred to a PVDF membrane, and bound anti-TLH scFv-LA3 was detected using an anti-E-tag HRP-conjugated antibody. (E) Binding of scFv-LA3 to different concentrations of TLH antigen as determined by ELISA. The four TLH concentrations were 5 μg/ml (A), 2.5 μg/ml (B), 1.25 μg/ml (C), and 0.625 μg/ml (D).

Fig 6

Neutralizing activity of scFv-LA3. (A) The neutralizing activity of scFv-LA3 for TLH-induced cell toxicity is dose dependent. HeLa, Changliver, and RAW264.7 cells were cultured in triplicate in 96-well flat-bottom plates and treated with 10 μg/ml of TLH, with or without various dilutions of phage scFv-LA3. Cell growth was evaluated using the MTT assay. (B) Time course of scFv-LA3 neutralization. HeLa, Changliver, and RAW264.7 cells were cultured in triplicate in 96-well flat-bottom plates and then treated with 10 μg/ml of TLH and the same concentration of phage scFv-LA3 (10¹⁰ CFU/ml) for different lengths of time (at intervals of 3 h) for up to 27 h. Cell growth was evaluated using the MTT assay. Data are presented as means ± SD for three independent experiments. (C) FACS analysis. Changliver and RAW264.7 cells were cultured in triplicate in 6-well flat-bottom plates and treated with 5 μg/ml of TLH and the same concentration of phage scFv-LA3 (10¹⁰ CFU/ml). Cells were incubated for 24 h and stained with FITC-conjugated annexin and propidium iodide.