Expression, purification, and identification of α-HL
The cDNA of α-HL was obtained by reverse transcription using total RNA as the template and Oligo dT15 as primers. The fragment of the α-HL gene was amplified using nested PCR. The results showed that the size of the α-HL gene fragment was about 900 bp (Fig. 1A). The soluble α-HL fusion protein was expressed with IPTG induction at low temperatures. The expression of the α-HL fusion protein was detected by western blot analysis, and the expressed protein was confirmed to be the target protein α-HL (Fig. 1B). After purification, the results of SDS-PAGE showed that the target α-HL fusion protein was purified successfully, and its size was about 90 kDa (Fig. 1C). In the N-terminal of expressed α-HL, a trigger factor of 48 kDa was connected, which was mainly used to increase the solubility of protein expression.
Preliminary screening of fully human scFvs against α-HL
The purified α-HL was used for phage screening. Specific single-chain antibodies were enriched after four rounds of phage display. More than 1000 clones were randomly selected by enzyme-linked immunosorbent assay (ELISA). The results showed that some clones had better binding reactions with the antigen α-HL, in which the positive scFvs with OD450 > 0.8 accounted for about 34%. Eighty scFv clones were randomly selected for expression, and the binding between α-HL and scFvs was detected by ELISA (Fig. 2).
Expression and identification of scFvs against α-HL
The scFvs gene with the highest ELISA OD values was inserted into the prokaryotic expression vector pLZ16 for soluble expression. The binding activity and specificity of expressed scFvs to S. aureus were determined by ELISA. The results showed that 15 scFvs could specifically bind to S. aureus, but not to S. albicans (Fig. 3A). The 15 scFvs were identified by western blot analysis, and the scFv protein was the target protein with a size of 30 kDa (Fig. 3B). The 15 scFvs also could bind well to the α-HL of S. aureus (Sigma, USA). (Fig 3C). The sequencing results showed that the sequences of 15 scFvs were correct, all of which were open reading frames, and their CDR regions were different (Fig. 3F).
Construction, expression, and purification of scFv-Fcs
The toxicity test of α-HL on A549 showed that three scFvs (scFv10, scFv555, and scFv802) showed better neutralizing activity. Due to the short half-life of scFv in vivo, the Fc segment was added to the three scFvs that performed well in the hemolysis blocking experiment, thus generating an IgG-like scFv-Fc antibody. The SP-scFv-Fc/pcDNA3.1 and SP-scFv-Fc/pMH3 recombinant vectors were constructed to examine the eukaryotic expression of soluble scFv-Fcs (Fig. 4A and 4B). The three scFv-Fcs were expressed in pcDNA3.1 and pMH3. Western blot results showed that the size of scFv-Fcs was about 55 kDa, and the expression level of scFv-Fcs in PMH3 was significantly higher than that in pcDNA3.1(Fig. 5A). The purified expressed α-HL (HLA-EX) and the commercial hemolysin (HLA-Sigma) were used as antigens in ELISA. The result showed that the expressed proteins could bind well to α-HL (Fig. 5B and 5C). The three scFv-Fcs were highly expressed in the pMH3 vector and were purified (Fig. 4D) for subsequent functional verification of scFv-Fcs.
Functional verification of scFv-Fcs
The neutralization properties of the three purified scFv-Fcs were further verified. A549 cytotoxicity tests showed that all three scFv-Fcs could neutralize cytotoxicity, reduce cytotoxicity, and protect A549 cells to varying degrees compared with the control group. Among these, scFv555-Fc has a better neutralizing effect than the other two. (Fig. 6A). The results of the anti-rabbit hemolysis test showed that scFv555-Fc had the most obvious anti-hemolytic effect (Fig. 6B). The results show that the three scFv-Fcs screened had a certain neutralization effect on hemolysin.
Establishment and evaluation of the scFv555 homologous model
The Discovery Studio 2016 software predicted CDRs of the scFv555 antibody. A total of 25 templates were obtained using the “identify framework templates” module. 4M6O LH was selected as the overall template, 4R4B A as the light chain template, and 3NH7 H as the heavy-chain template. The scFv555 model, including the frame region and the loop region, was constructed. Then, scFv555-M0019 was selected as the best 3D model using PDF total energy, PDF physical energy, and DOPE score (Fig. 7A). The Ramachandran diagram was used to evaluate the model scFv555-M0019. The results showed that more than 90% of the amino acid residues fell in the allowable region (purple) and the optimal region (blue), indicating that this model conformation conformed to the rules of stereochemistry (Fig. 7B). The profile-3D image showed that most of the Verify Score was > 0. The scFv555-M0019 was matched with its own amino acid sequence, and therefore the model had great credibility (Fig. 7C).
Antibody–antigen docking and key residue analysis
The scFv555 model as the receptor and α-hemolysin monomer from the MEDI4893·AT complex (PDB code: 4U6V) as the ligand were docked using the ZDOCK module. A total of 26 poses were chosen using the ZDOCK score (ZDOCK score > 20) and the spatial structure to optimize RDOCK. The best E_RDOCK score for docking was pose 2 (Fig. 8A). Then the “analysis protein interface” module was used. The analysis results suggested that the key amino acid residues of interface prompt for α-hemolysin were VAL26, TYR28, HIS35, LYS37, PHE39, ARG56, LYS58, THR60, HIS144, ASN214, and GLY223 (Fig. 8B). Meanwhile, the binding region of scFv555-M0019 was mainly located in the heavy-chain CDR3 region.
In the meantime, the scFv555 model as the receptor and α-hemolysin monomer from the LTM14·AT complex (PDB code: 4IDJ) as the ligand were docked using the ZDOCK module. A total of 23 poses were chosen using the ZDOCK score (ZDOCK score > 20) and the spatial structure to optimize RDOCK. The best E_RDOCK score for docking was pose 6 (Fig. 8C). Then, the analysis interface module was used. The analysis revealed that the key binding sites of the α-hemolysin monomer for this binding model were ILE7, TYR28, LYS37, PHE39, ARG56, LYS58, ILE142, LYS147, and SER222 (Fig. 8D). Meanwhile, the binding region of scFv555-M0019 was mainly located in the heavy-chain CDR3 region.
The two α-hemolysin monomer models from different complex crystals (PDB code:4U6V and 4IDJ)were used to simulate the antigen-antibody binding with the model of scFv555 , and it was found that some key binding sites (TYR28, LYS37, PHE39, ARG56, and LYS58) of α-hemolysin bound to scFv555 model could be overlapped.