Vectors, Bacterial Strains, Helper Phage, and Libraries
The cloning and expression vector pET-Sumo, E. coli strain BL21 (DE3) and SUMO enzyme were purchased from Invitrogen Biotechnology Co. (CA, USA). The pET28a-TAT-GFP vector was constructed previously by our research group. A Tomlinson (I+J) phage antibody library, E. coli strains (TG, HB2151) and helper phage (KM13) were obtained from the Medical Research Council in Cambridge, UK. Goat anti-mouse IgG labeled horseradish peroxidase (HRP) antibody and anti-HIS6 labeled antibody were purchased from Abbot Trading Co., Ltd (Shanghai, China). The Protein A-HPR antibody was purchased from Abcam Co. (Cambridge, England). SP Sepharose 4 FF, Chelated Sepharose 4 FF, rProtein-A Sepharose 4 FF, and AKTA Prime chromatographs, were purchased from GE Healthcare (Fairfield, CT, USA). Stocks of H5N1 (A/Meerkat/Shanghai/SH-1/2012) and H1N1 (A/Changchun/01/2009) viruses are held in our Institute.
Expression and Purification of H5N1-M1 Protein
The cDNA sequence of the M1 protein for H5N1 virus was identified in the NCBI library; this sequence was then used as a template for primer design: forward: 5′-ATG AGT CTT CTA ACC GAG GTC-3′ and reverse: 5′-CCG GAA TTC TTA CTT GAA TCG CTG CAT CTG CAC T-3′. The M1 gene was amplified using H5N1 (A/Meerkat/Shanghai/SH-1/2012) cDNA as a template. The PCR reaction conditions were as follows: 94°C denaturation for 5 min, 94°C for 50 s, 50°C for 50 s, 72°C for 50 s (for 34 cycles) followed by 72°C 10 min. The amplification products were separated by gel electrophoresis and then ligated into the PET-SUMO vector with T4 ligase. The vector was sequenced and then transformed into BL21 (DE3) competent cells.
Biopanning of a Phage Display Library and Selection by M1 specificity
The phage antibody library was prepared in accordance with the manufacturer's instructions and then screened with the purified M1 protein as an antigen. During this process, the M1 protein was coated in 96-well plates (Nunc-Nalgene, USA) at a concentration of 5µg/ well and then incubated at 4°C overnight. The next day, the supernatant was discarded and washed three times with PBS to remove the non-adsorbed antigen. Non-specific binding was blocked with 200 µL of 2% milk/PBS at 37°C for 2 hours. After discarding the sealing solution, the wells were washed three times with PBS, and the liquid was removed by vigorous shaking. Next, the Tomlison I+J phage antibody library was added and diluted with 2% milk/PBS to a titer of 1.0 ×1013; 100 µL was added to each well, and the liquid was incubated with vigorous shaking at room temperature for 60 min. After standing for 60 min, the liquid was discarded, and the wells were washed 10 times with PBS (0.05% (V/V) Tween-20). The residual liquid in each well was patted dry and 50µL of eluting solution (5 mg/mL trypsin-PBS) was added to each well. The plates were then shaken at room temperature for 15 min to eluate the phages, which were then stored at 4°C. The eluded phage was cloned into E. coli TG1 and further panning was performed. Second, third, and fourth rounds of panning were performed under similar conditions, except that the concentration of the antigen coating was reduced to 2 µg/well. Unbound phages were removed by 20, 30, and 40 washes with PBS (0.05% (V/V) Tween-20).
Next, 2% milk/PBS (100µL/Well) was added to the plate, and the plate was kept overnight at 4℃. Nonspecific binding was then blocked with 2% BSA/PBS for 2 h and the phage antibody from the fourth round of screening was added. After incubation at room temperature for 1 h, the supernatant was collected to remove the phages that had been specifically adsorbed to the milk powder in the antibody library; the collected phages were then stored at 4°C.
Expression of Positive Clones and ELISA Analysis for M1 Protein
After four rounds of screening, 10µL of phages were added to 200µL of fresh E. coli HB 2151 and left for 30 min in a water bath at 37°C. Then, 50 µL was applied to a TYE (15 g bacto-agar, 8 g Nacl, 10 g tryptone, 100g Ampicillin, 10g Glucose, 5 g yeast extract in 1L) plate and cultured overnight at 37°C. Once grown on the plate, single colonies were randomly selected and placed on a 96-well culture plate; each well contained 100 µL of 2×TY (30 g bacto-agar, 16 g Nacl, 20 g tryptone, 100g Ampicillin, 10g Glucose, 10 g yeast extract in 1L) medium and cultured overnight at 37°C. The next day, approximately 2 µL of bacterial solution from each well was placed in another 96-well cell plate (the remaining solution was added to glycerol at a final concentration of 15% and stored at -70°C). The new cell plate contained 200µL of 2×TY medium (containing 100 µg/mL Amp and 0.1% glucose) from each well and cultured at 37°C to an OD600 of 0.9 (after approximately 4 h of culture). Isopropyl β-D-Thiogalactoside (IPTG) at a final concentration of 1 mmol/L was added to each well and cultured overnight on a 30°C shaker. After overnight culture, the bacterial solution was centrifuged at 1,800 g for 15 min; the supernatant was then transferred to a new plate and stored at 4°C to await testing.
M1 protein (2 µg, 100 µL/well) was added to 96-well plates and incubated overnight at 4°C. The next day, the plates were washed three times (3 min each time) with wash solution (0.05% PBS (V/V) and Tween-20). Next, 200 µL of 2% milk/PBS was added to each well and incubated at 37°C for 1 h. Then, 100 µL of HB2151-induced supernatant was used as a negative control; this was added to each well and incubated at 37°C for 1h. Next, the enzyme label plate was washed three times (for 3min each time) and the excess liquid was patted dry. Next, 100 µL (1:500) of Protein A-HRP was added to each well and incubated at 37°C for 1h. Washing was carried out three more times (3min each time) and excess liquid was patted dry. o-Phenylenediamine (OPD) solution (100 µL) was then added to each well and incubated at room temperature in the light for 20 min. Finally, 2 mol/L of sulfuric acid (50 µL) was added to each well to stop the reaction and the OD490 absorption value was determined.
Sequence Determination of Selected Phage Clones and the Expression & Purification of HuScFv
The M1-positive binding phage in the monoclonal ELISA was used as a template, and vector specific primers (LMB3: 5ʹ-CAG GAA ACA GCT ATG AC-3ʹ; PHEN: 5ʹ-CTA TGC GGC CCC ATT CA-3ʹ) were used to amplify the HuScFv gene fragment. The obtained amplification products were subsequently detected by 1% agarose gel electrophoresis. The PCR amplification conditions were as follows: pre-denaturation at 94°C for 5 min, 94°C for 50 s, 54°C for 50 s, 72°C for 120 s (35 cycles) and a final extension at 72°C for 10 min. The target DNA fragment was then recovered and sequenced by Kumei Biological Engineering Co. (China).
ELISA-positive strains were transferred to 5 mL of 2× TY medium containing 100 µg/mL Amp and 1% glucose and cultured overnight at 37°C. The next day, 200 µL of overnight culture was transferred to 2× TY medium (containing 100 µg/mL Amp and 0.1% glucose) and cultured at 37°C to an OD600 of 0.9 (approximately 4 h). A final concentration of 1 mmol/L of IPTG was added for overnight induction on a shaking table at a 30°C. On the third day, the induced bacterial solution was centrifuged at 5000 g (Beckman, USA) for 30 min; the supernatant was removed and precipitated with 10%–55% saturated ammonium sulfate. The precipitated solution was then resuspended with 30 mmol/L of PB (pH7.2) and dialysis was performed in PBS overnight. The crude samples were then purified by Protein-A FF affinity chromatography; eluted samples were dialyzed with PBS overnight. The target protein was finally analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Western Blotting & Immunoaffinity Analysis with HuScFv
The purified M1 protein was separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane using a protein electrophoresis transfer device (BIO-RAD) at 45 V for 35min. Membranes were then blotted with HuScFv as a primary antibody and ProteinA-HRP as a secondary antibody (diluted with 1:2,000 PBS). DAB was used as a color reagent to detect the immunobinding activity of the antibody. The binding ability of HuScFv to M1 was determined by a non-competitive ELISA method. Different concentrations of the M1 protein antigen were coated with skimmed milk powder for 1 h, and different dilutions of HuScFv were added. Then, proteinA-HRP and OPD color solution were added; 2 M of sulfuric acid was used as a termination solution, and the absorbance was measured at 490 nm wavelength. Affinity constants were calculated using the formula Ka . IgBLAST (a tool for immunoglobulin (IG) and T cell receptor (TR) V domain sequences, NCBI) was used to analyze the nucleotide sequences of clones with the highest immune affinity constant to determine the HuScFv framework and complementary determination region (CDR).
Expression and Purification of TAT-HuScFv
Forward (5'-GTG AAT TCA TAA TGA AAT ACC TAT TGC CT-3') and reverse (5 '-GCA AGC TTC TAT GCG GCC CCA TTC AG-3') sequences were introduced into EcoR I and Hind III sites, respectively. A HuScFv plasmid with a high immunoaffinity constant was extracted and used as a template for PCR. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min, then 34 cycles of 94°C for 50 s, 50°C for 50 s, and 72℃; this was followed by a final extension at 72°C for 10 min and cooling at 4°C. Amplification products were recovered by gel electrophoresis. The amplification products and the vector (pET28a-TAT-GFP) were extracted by the double digestion of EcoR I and Hind III, respectively; the amplification product was then ligated with T4 ligase and transformed into competent E. coli DH5α. Positive clones were then identified by PCR.
The pET28A-TAT-HuScFv construct was then transformed into BL21(DE3) with the CaCl2 method. A single colony was selected and inoculated into LB liquid medium (containing 50 μg/mL Kan) at 37°C and 180 rpm until the OD600 was approximately 0.5. IPTG was added to a final concentration of 1 mmol/L and induction was carried out over a culture period of 4 h. The induced bacterial culture was centrifuged at 4°C at 5000g for 20 min, and the bacteria were collected. The bacteria were suspended with TE (pH 8.0) buffer solution and ultrasonically crushed in an ice bath (power: 1500 W; working time: 5 s; interval time: 9 s; 50 times in total). Microscopic examination confirmed that the bacteria had been completely broken down. Centrifugation was carried out at 12,000 g; samples of supernatant and precipitate were then separated by 12% SDS-PAGE.
The supernatant of the expressed cells was obtained following ultrasonic lysis, and a metal chelated Cu2+ column was used as the buffer system for PBS (pH 7.2). The elution peaks of the target proteins were analyzed with 20 mM and 200 mM imidazole, respectively. The eluent was then purified on a rProteinA FF column. Finally, eluted samples were dialyzed with PBS.
Hemagglutination Inhibition Analysis of Anti-M1-HuScFv and TAT-HuScFv
Digested MDCK cells were placed in a 96-well cell culture plate (3×104 cells/well), and the cells grown into a single layer to be absorbed into the medium. The cells were washed three times with DMEM, and A/Changchun/01/2009 H1N1 was added to each well at different concentrations (PBS was added to the negative control well). The cells were incubated at 37°C for 3.5 h, and the extracellular fluid was discarded. Cells were washed twice with PBS; purified HuScFv and TAT-HuScFv (10 μg/well; the positive control included PBS only) were then added and incubated at 37°C for 1.5 h. The supernatant of cells was then absorbed and DMEM (containing 1% FBS) was added to each well and cultured overnight at 37°C with 5% CO2. The next day, the hemagglutination inhibition test was performed with the overnight culture supernatant. The supernatant was added to the reaction plate (50 μL/well), along with fresh 0.85% chicken red blood cell suspension (50 μL/well), and incubated at room temperature for 30 min; the results of the test were observed by the upright reaction plate.
TAT-HuScFv Bound to Amino Acid Sites of M1 Protein Epitopes
The M1 protein was sequenced and then decomposed in order from the N-terminal to the C-terminal to synthesize 10 polypeptides (Table 1). Positive fragments were detected by the sandwich ELISA method, as described earlier. The polypeptides were used to coat 96-well plates, using TAT-HuScFv as the first antibody, and protein A-HRP as the second antibody. Analysis involved 3, 3′,5 ,5′-Tetramethylbenzidine (TMB) color and the positive fragments. The positive fragments were decomposed into peptides according to the overlapping sequences of four amino acids and then detected by sandwich ELISA. Positive results were analyzed and positive fragments were synthesized into peptides; these were tested again by sandwich ELISA until the amino acid sites that bound to TAT-HuScFv were identified.