Development of anti-aflatoxin B1 nanobodies from a novel mutagenesis-derived synthetic library for traditional Chinese medicine and foods safety testing

Cell culture

The normal human embryonic liver cell line CL48 was cultured in Eagle’s minimum essential medium supplemented with 10% fetal bovine serum. The human hepatoblastoma cell line HepG2.2.15 derived from the hepatocellular carcinoma cell line HepG2 with stable expression of transfected hepatitis B virus was cultured in Eagle’s minimum essential medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 1% nonessential amino acid, and 1% sodium pyruvate. The cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). The cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.

Synthetic nanobody library construction

Phage display libraries were constructed following the oligonucleotide-directed mutagenesis procedure proposed by Kunkel [22]. For more detailed descriptions, please refer to the protocol developed by Sidhu and Weiss [23]. In brief, this study employed structural biology and computer-aided mining to select a released nanobody structural sequence as the template (derived from protein data bank [PDB]: 3QXV). The three-dimensional template had a glove-like concave configuration, which inspired us to design a novel synthetic phage-displayed nanobody Golden Glove (SynaGG) library. PDB: 3QXV is a llama CDR1-4 graft nanobody antibody in complex with MTX. We synthesized a template nanobody gene harbouring TAA stop codons at positions chosen for randomization and subcloned it into the pCANTAB5E phagemid (GE Healthcare Inc.). Then NNK degenerate primers were used to mutate codons specifying 8 positions on template nanobody CDRs or FRs. TAA stop codons strategically positioned on the parent phagemid ensured that only phagemids carrying degenerate codons would yield fusion proteins with the pIII protein on the bacteriophage surface. To prevent interference from the degenerate NNK-producing TAG stop codon, Escherichia coli strain ER2738 (a supE strain with glutamine-inserting amber (UAG) suppressor tRNA) was employed for library amplification. TAG stop codons are suppressed by glutamine in ER2738. Thus, we transformed the constructed library DNA into the ER2738 through electroporation and calculated the transformed numbers to estimate the complexity of the library. The VCS-M13 (Stratagene Corp.) helper phage was added to the transformed culture to initiate recombinant phage production. The phage library was precipitated with 4% polyethyl glycol 8000 and 3% NaCl (w/v), resuspended in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA), and stored at 4 °C.

Biopanning and nanobody expression

After constructing the synthetic nanobody library, we used a panning method to enrich and isolate nanobodies with the specific ability to bind with AFB1 in the library. First, an AFB1 and BSA conjugate (AFB1-BSA) purchased form Sigma-Aldrich Corporation was coated onto the wells of a microtiter plate at 4 °C overnight. AFB1-BSA of 5 μg/well was used as the coating for the first round of panning; the antigen coating concentration in the second to fourth rounds was lowered to AFB1-BSA of 1 μg/well. The next day, the AFB1-BSA was removed, and the well was blocked with 3% BSA at room temperature for 1 h. Subsequently, library phage particles (1011) were mixed with 3% BSA at a 1:1 ratio, and this mixture was then added to the well and incubated at room temperature for 2 h. Next, unbound phages were removed, and the well was washed 10 times through pipetting with PBS with 0.05% Tween 20 (PBST). Bound phages were eluted with 0.1 M HCl–glycine (pH 2.2)/0.1% BSA elution buffer and neutralized with 2 M Tris base buffer. The eluted phages were then used to infect E. coli strain ER2738 immediately for phage amplification. The amplified phages were precipitated and recovered according to a previously described method and used in the next round of panning [24]. The panning procedure was repeated four times. After panning, total library DNA was purified and transformed into E. coli strain TOP 10F′ (a nonsuppressor strain; Invitrogen). The transformed clones were randomly selected for individual nanobody expression and subsequently for binding analysis by ELISA. The positive clones were sequenced to infer the nanobodies’ primary structure. The nanobody genes were fused with HA and His tags. For further nanobody protein expression and purification, the interesting clone was grown overnight in 0.5 mM isopropyl b-D-thiogalactopyranoside (IPTG) for nanobody induction. Subsequently, recombinant nanobodies were purified with Ni2+-charged sepharose according to the manufacturer’s instructions (GE Healthcare Inc.).

Sequence analysis

To sequence the nanobody clones of interest, we used a primer (5′-GCTATGACCATGATTACGCCA-3′) complementary to the pectate lyase B signal sequence placed before the heavy chain variable region. Next, the international ImMunoGeneTics (IMGT) information system/V-QUEry and standardization system (http://imgt.org) were used to compile and analyse the sequence data.

Enzyme-linked immunosorbent assay

AFB1-BSA, MTX-BSA, or AFB1–ovalbumin (OVA) (0.5 μg/well) was coated onto the wells of a microtiter plate at 4 °C overnight. These wells were blocked with 5% skimmed milk, and nanobodies were then added to the wells at room temperature for 1 h. After a wash with PBST, bound nanobodies were detected, and signals were developed using the horseradish peroxidase (HRP)–conjugated mouse anti-HA tag antibody (Cell Signaling Technology, Inc.). Finally, 3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB) was added for signal development. The reaction was stopped through the addition of 1 N HCl, and absorbance was determined through optical density (OD) measurement at 450 nm.

Competitive inhibition assay

Competitive inhibition assays were employed to determine the binding specificity of AFB1-binding nanobody molecules. In brief, microtiter plates were coated with AFB1-OVA at 0.5 μg/well and blocked with 5% skimmed milk. After 1 h of incubation at room temperature, the plates were washed with PBST. The purified nanobody of 10 μg/mL was incubated first with multiple concentrations of soluble free AFB1 diluted in ddH2O (ranging from 1.6 to 100 ng/mL) at room temperature for 1 h. Then, the mixtures were added to the wells to react with the coated AFB1-OVA molecule. After incubation at room temperature for 1 h, the plates were washed with PBST. Subsequently, the HRP-conjugated mouse anti-HA tag antibody (Cell Signaling Technology, Inc.) was added at room temperature for 1 h to detect bound nanobodies. After a wash with PBST, TMB substrate was added to each well for development. The reaction was stopped with 1 N HCl, and signal intensity was measured through OD measurement at 450 nm.

Cell proliferation assay

Cell proliferation was measured using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Cell Proliferation Assay Kit (Promega). HepG2.2.15 or CL48 cells were seeded at a density of 5000 cells/well in a 96-well culture plate. AFB1 was mixed with or without different concentrations of nanobodies and incubated at room temperature for 30 min. The mixture was then added to the cell culture and incubated for 48 h. Finally, an MTS and phenazine methosulphate mixture was added and incubated for 90 min for development. After the sodium dodecyl sulphate (SDS) reagent was added to stop the reaction, the absorbance of each well was determined through OD measurement at 490 nm.

Molecule modelling and molecular docking

To investigate how the candidate nanobodies A1, F2, and A1F2 interacted with AFB1, homology modelling was employed to create a three-dimensional structure of the nanobody with PDB: 3QXV as the template in BIOVIA Discovery Studio (Dassault Systèmes, BIOVIA Corp., San Diego, CA, USA). The crystal structure of the template VHH antibody was downloaded from the Research Collaboratory for Structural Bioinformatics Protein Data Bank. The interaction between proteins and the AFB1 compound was simulated using the CHARMm force field. The AFB1 compound was docked into the CDR-H loop of the nanobody with the GOLD docking tool. The docking parameter settings are described as follows: The MTX structure of the VHH template (PDB: 3QXV) was set as the centre to define the binding site sphere (10 Å). The GOLD tool with a genetic algorithm was used to simulate AFB1 compound docking into the CDR pocket of a nanobody with a flexible state. In total, 200 docking runs were generated for each AFB1 molecule to simulate the interaction of AFB1 with the CDR binding site of the nanobody. These conformations were then further subjected to automatic methods to identify the optimal AFB1–nanobody interaction configurations. During the simulation, the cation-pi interaction, hydrophobic interaction, and ligand torsion strain were accounted for in the CHEMPLP score function.

Statistical analysis

All data were analysed using GraphPad Prism (GraphPad, CA, USA) and are presented as mean ± standard error of the mean. Statistical comparisons between groups were performed using a one-way analysis of variance followed by post hoc analyses with the Tukey HSD protected least significant difference test. P values lower than 0.05 were considered significant.

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