Bactrian camel immunization
A healthy 3-year-old female Bactrian camel was selected for immunization with adenovirus rAd26 and ChAd63 (gifted by Professor Jinsheng He from Beijing Jiaotong University). The adenoviruses were diluted from the original concentration of 1×1013 vp/ml to 2×1011 vp/ml to 1×109 vp/ml for the immunization. 5 ml of blood was collected from the jugular vein and left to stand until it coagulated naturally before immunization. The serum was taken and stored at -20°C. The camel was immunized four times, and the interval between immunizations was two weeks. A week after each immunization, blood was collected from the jugular vein, and placed at 4°C to separate the serum used for ELISA assay to detect antibody titer. Two weeks after the fourth immunization, 50 ml of blood was collected from the jugular vein of the immunized camel for the separation of peripheral blood lymphocyte. The camel was farmed in the pasture that located in the suburb of Hohhot city, Inner Mongolia, and was provided free access to water and food. All experimental procedures were performed in accordance with the institutional and national guidelines and regulations and were approved by the Animal Care and Use Committee of Inner Mongolia Agriculture University.
Construction and screening of Bactrian camel phage display antibody library
Construction and screening of single domain phage display library as shown in Fig.1. The peripheral blood monocyte (PBMC) was prepared by the test method of the animal blood lymphocyte separation kit (TBD science, purchased from Tianjin Haoyang Biological Products Technology Co. Ltd.). Using TRIzol reagent (Ambion, USA) to extract total RNA from PBMC, and reverse transcripted to cDNA with ted reverse transcription kit (Promega, USA) by using random hexamer as primer. The VHH fragment was amplified by the nested PCR method using the primers as listed in Table 1. In the first round of PCR, using P1 and P2 as primers, the antibody gene sequence from leader peptide to CH2 region was amplified from the cDNA. The PCR products were run on agarose gel, and the VHH containing band (600 bp) was recovered by cutting and extracting from the gel and used as the template for the second round of PCR. The full-length VHH gene from FR1 to FR4 with a fragment size of about 400 bp was amplified from the second round of PCR using P3 and P4 as primers. The PCR amplified product was digested with restriction enzymes NcoⅠand NotⅠ (New England Biolabs, UK) and ligated into the pMECS plasmid vector (a gift from Professor Serge Muydermans of the Vrije Universiteit Brussel, Belgium) with T4 ligase (New England Biolabs, UK), and transformed the ligation product into E. coli TG1 competent cells (GE Healthcare, USA) to construct the antibody library. Estimate the capacity of the antibody library by calculating the colony forming units (CFU) of 10 series of multiple dilutions on a 2×YT-AG plate. The transformation efficiency and the insertion of VHH was evaluated by PCR method with the sequencing primers of pMECS plasmid vector MP57 and GIII as listed in Table 1.
The antibody library was added to 200 ml of 2×YT medium and the OD600nm value of the bacterial solution does not exceed 0.3, then culture the library in a shaker at 37 °C, 250 r/min for 2 h; Added M13K07 helper phage (GE Healthcare, USA) and incubated at 37 °C for 1 h for infection; The cultured bacteria solution was centrifuged at 2 000×g for 10 min at 4 °C; discard the supernatant, resuspended, and inoculated the solution into 2×YT-AK medium containing ampicillin and kanamycin at 37 °C, 220 r/min shaking overnight; The cultured medium was centrifuged at 4°C, 7197×g for 15 min in the next day, and take the supernatant, then add 20% PEG8000 and centrifuge at 7197×g at 4°C for 25 min to precipitate the phage; Discarded the supernatant, and resuspended the pellet in PBS to obtain the recombinant phage library. Adding the phage library to the 96-well microtiter plate coated with the adenovirus rAd26 at a concentration of 2×107 vp/ml, and binding at 37°C for 1h; eluting the bound phage with 100 μl 100 mM triethylamine, sealing, incubating at room temperature for 10 min, and neutralizing with 50 μl 1 M Tris-HCl. The eluted recombinant phage was used to infect E. coli TG1 (OD600nm = 0.6), and M13K07 helper phage was added for rescue. The phages were harvested, purified and used for a new round of enrichment. The E. coli TG1 infected with the eluted phages from the final enrichment were grown on 2×YT-AG plates. 92 clones were randomly picked from the 2×YT-AG plate and added them to the 2×YT-AG liquid medium, incubated at 37°C, 250 r/min overnight. The next day, the M13K07 helper phage was added and infected for 1 h; after centrifugation at 14000 r/min for 5 min, the bacteria were resuspended in 2×YT-AK medium, and incubated at 37°C, 250 r/min overnight. The medium was centrifuged at 14000 r/min for 5 min in the next day, and the supernatant was collected, 20% PEG8000 was added, and the mixture was centrifuged at 7197 × g for 20 min. The recombinant phage was added to an ELISA plate pre-coated with a concentration of 2×107 vp/ml adenovirus rAd26, and the M13K07 helper phage was used as a negative control, PBS buffer was used as a blank control, and binding for 2 h at room temperature; HRP/Anti-M13 Monoclonal Conjugate (GE Healthcare, USA) diluted at 1:5000 and added to each well, binding for 1 h at room temperature; TMB solution was added for color development, and determine the OD450nm value. The absorbance of the experimental group/negative control≥2.1 was regarded as positive.
Expression and purification of the sdAb
The plasmids of the positive clones were isolated from the phage-ELISA, and double digested with restriction enzymes NcoⅠand NotⅠ. The digested VHH fragments were ligated into the pET-22b (+) –SBP (The pET-22b (+) vector carried a streptavidin binding protein that constructed and preserved by the Public Health Department of the School of Veterinary Medicine, Inner Mongolia Agricultural University) plasmid that digested by the same restriction endonuclease with T4 DNA ligase. The products were transformed to E. coli BL21 (DE3) competent cells (TransGen Biotech, Beijing, China). The expression of recombinant sdAb should be induced by IPTG (Solarbio Life Sciences, Beijing, China) for 8-12 h. Then the cells were collected and sonicated. Afterwards, the precipitate and supernatant were collected and analyzed by SDS-PAGE. Ni-NTA Sefinose™Resin (Sangon Biotech, ShangHai, China) was used to purify the expressed recombinant single domain antibodies, and the purified sdAbs were analyzed by SDS-PAGE.
Binding activity and specificity of the sdAb
In order to determine the binding activity of the purified recombinant sdAbs, the purified clone 5 and 31 sdAbs were diluted and added as a primary antibody to a 96-well plate coated with adenovirus rAd26, ChAd63 and HAd5 at a concentration of 2×107 vp/ml. At the same time, the sonicated E. coli BL21 (DE3) that transformed with empty pET-22b (+) vector was used as negative control, and PBS buffer was used as blank control. The HRP-conjugated 6*His-tag Mouse monoclonal antibody (Proteintech Group, WuHan, China) was used as the secondary antibody at a concentration of 1:10000, and after added the TMB solution for color development, and determine the OD450nm value on microplate reader.
One microliter per well of polylysine was added to a 24-well cell culture dish, and incubated for 10 min, discarded the liquid, and dry in the super clean bench. HEK293A cells were incubated with a 105 cells/well in a 24-well cell culture dish at 37°C and 5% CO2 for about 24 h; the cell culture medium was discarded, and added 1 ml of DMEM medium to each well; the adenovirus rAd26 was diluted to an concentration of 2×107 vp/ml, and added 100 μl of virus diluent to each well, and added DMEM to the control group, and cultured in an incubator for 24 h; the medium was discarded and added 1 ml of paraformaldehyde to each well to fixing for 20 min at room temperature, then aspirating the fixative, and rinsed with PBS 3 times for 5 min each time. Adding 100 μl 0.3% TritonX-100 dropwise to each well, and incubated at room temperature for 20 min, and rinsed with PBS 3 times, each time for 5 min. Adding 100 μl 5% BSA solution to each well, and incubated at room temperature for 30 min, prepared the primary antibody working solution with PBS to the concentration of 200 μg/ml, and added 100 μl to each well, and incubated overnight in a refrigerator. The 24-well cell culture dish was taken out of the refrigerator, placed at room temperature to rewarm for 10 min, and PBST was added to the shaker and rinsed 3 times, each for 5 min. The CoraLite®488-conjugated 6*His His-Tag Mouse Monoclonal antibody (Proteintech Group, WuHan, China) was used as the secondary antibody at a concentration of 1:150, and added 100 μl of the working solution of the secondary antibody, incubated at room temperature for 1 h, added PBST to the shaker and rinse 3 times, each time for 5 min. 100 μl ready-to-use DAPI working solution was added and incubated at room temperature for 4 min, and rinsed with PBS 3 times, 5 min each time. The fluorescence signal was detected by a confocal microscope (ZEISS, LSM-800).
Immunoaffinity purification of adenovirus from cell culture medium with immobilized sdAb
The conjugation of sdAb to the NHS-Activated Sefinose™ 4 Fast Flow (BBI, Beijing, China) was done according the manufactures instruction. One microgram of purified sdAb was dissolved in 1 ml of coupling buffer (NaHCO3 and NaCl solution). One microliter of NHS-Sepharose FF was added into the gravity column, and take 5 mL of 1 mM HCl to wash the column three times to remove the preservation solution, and added antibody solution immediately after washing, and shake overnight at 4°C for better coupling. The filtrate was collected in the next day, and rinsed the coupled sepharose with 5 ml coupling buffer, and use 1 ml of blocking buffer to shake at room temperature for 4 h to block uncoupled agents. After removing the blocking buffer, the sepharose was washed three times with 2 ml of coupling buffer. The recombinant human adenovirus HAd5 expressing enhanced green florescent protein (EGFP) (prepared in Department of Veterinary Public Health Faculty) was diluted in cell culture medium with the titer of 107 vp/ml, and added to the prepared immunoaffinity column, and combined for 30 min at room temperature. The bound virus was eluted with 2 M and 4 M NaCl solution, and the eluent was added to the cultured HEK293A cell for reinfection. The presence of the recombinant virus in the eluent and in the reinfected cell was visualized under the confocal microscope (ZEISS, LSM-800) by the expression of the EGFP protein.