Fig 1.
Construction and identification of a BVDV nanobody library.
(A) Antibody titer was detected by agar diffusion test. Center bore: inactivated BVDV solution; NC: negative control. Order of antibody dilution set: 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256. (B) Nested PCR amplification results of VHH and the 450 bp sequences that were obtained. (C) Results of enzyme digestion and identification of pCANTAB5E-VHH M: Marker; 1–2: pCANTAB5E-VHH digested with SfiI. (D) The size of the library (1.3×1011 CFU/ml) was determined by counting the number of clones after gradient dilution.
Fig 2.
SDS–PAGE analysis of the expression and purification of BVDV-E2 recombinant protein.
Expression of BVDV-E2 in E. coli BL21 (DE3). When DE3 bacteria reached the logarithmic growth phase, IPTG was added to a final concentration of 1 mmol/l, and induced for 16h at 28°C and Western-blot results showed that the strip with the expected protein size of 49.5 kDa was consistent with Lanes 1 and 2 and purification of recombinant BVDV-E2 protein. Lane 3: The Western-blot results of BVDV-E2 protein. Lane M: Molecular weight markers, size indicated in kDa.
Table 1.
Selective enrichment of nanobodies from the libraries during panning.
Fig 3.
A total of 96 random clones from the library were analyzed with monoclonal phage ELISA. BVDV-E2 antigens at 10 μg/ml were coated in each well. PBS served as the negative control and M13K07 was used as the positive control. A total of nine clones were selected on the basis of absorbance. The x-axis shows the number of clones, and the y-axis shows the absorbance values at 450 nm.
Fig 4.
A total of nine clones (out of 96) were analyzed with monoclonal phage ELISA. BVDV-E2 antigens at 10 μg/ml were coated in each well. PBS served as the negative control. A total of five clones were selected on the basis of absorbance. The x-axis presents the clone number, and the y-axis shows the absorbance values at 450 nm.
Fig 5.
Multiple amino acid sequence alignment of BVDV-E2 nanobody clones.
The framework and CDR regions and amino acid numbering were performed as stipulated in Gene.DOC. The CDR regions outlined in lines. The CDR regions are outlined in lines. Sequencing analysis indicated that the nanobody clones were highly homologous to the camel VHH sequence.
Fig 6.
Purification of BVDV-specific nanobody.
The nanobody that was encoded by candidate DNA sequence was purified using affinity chromatography. The nanobody was detected using Coomassie brilliant blue staining under SDS-PAGE.
Fig 7.
Verification of the binding ability of the nanobody and antigen protein.
The optimal nanobody dilution ratio was determined by double antibody sandwich ELISA. When the dilution ratio of antigen was 1:20 and the dilution ratio of nanobody was 1:160 and the binding capacity was the strongest.
Fig 8.
Number of plaques in the field of view.
(A) Non-viral plaque of MDBK cells in the uninfected group; (B) BVDV concentrate and nanobody VHH15 were mixed thoroughly in a 15 ml centrifuge tube at a 2:1 ratio and incubated in a 37°C incubator for 60 min. After the nanobody VHH15 was incubated with BVDV, it was added to the MDBK cells, and viral plaques lessened; (C) BVDV concentrate and PBS were mixed thoroughly in a 15 ml centrifuge tube at a 2:1 ratio and incubated in a 37°C incubator for 60 min. BVDV-infected MDBK cells showed significant amounts of viral plaque.
Fig 9.
Effect of different doses of VHH nanobodies on BVDV replication copy number.
After different doses of nanobody and BVDV were prepared and incubation for 1.5–2 h, the mixture was used to infect MDBK cells. After 48 h and 72 h of incubation, the cells were collected and the total RNA was extracted and reverse-transcribed into cDNA, which was then subjected to qRT-PCR detection.