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Design and selection of a camelid single-chain antibody yeast two-hybrid library produced de novo for the cap protein of porcine circovirus type 2 (PCV2).

Fu X, Gao X, He S, Huang D, Zhang P, Wang X, Zhang S, Dang R, Yin S, Du E, Yang Z - PLoS ONE (2013)

Bottom Line: Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences.Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells.All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, China.

ABSTRACT
Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs) are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H) library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×10(6) cfu/3 µg and 2×10(9) cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

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Related in: MedlinePlus

Intracellular interaction of the selected VHHs with PCV2 Cap protein.The white clones in lane A indicate that the bait (pBD-Cap) has no autoactivator activity in yeast. When DNA-BD/Cap was cotransformed into Y2HGold yeast strain with 12 random positive AD/VHH plasmids, all co-transformants appeared blue on both DDO/X/A and QDO/X/A plates (Figure 4, lanes B-M). In the control group (Figure 4, lane N), the pGBKT7-53 and pGADT7-T co-transformants (positive control) appeared blue on both DDO/X/A and QDO/X/A plates, whereas pGBKT7-Lam and pGADT7-T co-transformants (negative control) appeared white on the DDO/X/A plate and did not grow on the QDO/X/A plate.
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pone-0056222-g004: Intracellular interaction of the selected VHHs with PCV2 Cap protein.The white clones in lane A indicate that the bait (pBD-Cap) has no autoactivator activity in yeast. When DNA-BD/Cap was cotransformed into Y2HGold yeast strain with 12 random positive AD/VHH plasmids, all co-transformants appeared blue on both DDO/X/A and QDO/X/A plates (Figure 4, lanes B-M). In the control group (Figure 4, lane N), the pGBKT7-53 and pGADT7-T co-transformants (positive control) appeared blue on both DDO/X/A and QDO/X/A plates, whereas pGBKT7-Lam and pGADT7-T co-transformants (negative control) appeared white on the DDO/X/A plate and did not grow on the QDO/X/A plate.

Mentions: Before Y2H experiments, we first confirmed that the BD-Cap fusion protein had no autoactivator activity in yeast (Figure 4, lane A). After mating the Y2HGold (pBD-Cap) and Y187 (pAD-VHHs) yeast libraries and incubating on DDO/X/A plates for 3–5 days, hundreds of yeast blue colonies were selected. However, only 36 clones were recovered after two more rounds of screening on QDO/X/A plates. The prey plasmids from the above 36 clones were isolated, recovered, and co-transformed with the bait plasmid (pBD-Cap) or pGBKT7 (empty insertion) to verify the positive interactions between VHHs and Cap protein in yeast strain Y2HGold. The co-transformation results showed that all 36 clones rescued from QDO/X/A plates were genuine positive clones. Lanes B-M of Figure 4 show 12 randomly selected positive clones. Meanwhile, both positive (pGBKT7-53 and pGADT7-T co-transformants) and negative (pGBKT7-Lam and pGADT7-T co-transformants) control groups were found eligible (Figure 4, lane N). The results for intracellular interaction between Cap and anti-Cap VHHs in yeast are also summarized in the Table S3 (Table S3 in File SI). Sequencing results showed that the 36 positive clones were of 21 different clonal origins and the insertions were homologous to the properties of camel VHH (Figure 5).


Design and selection of a camelid single-chain antibody yeast two-hybrid library produced de novo for the cap protein of porcine circovirus type 2 (PCV2).

Fu X, Gao X, He S, Huang D, Zhang P, Wang X, Zhang S, Dang R, Yin S, Du E, Yang Z - PLoS ONE (2013)

Intracellular interaction of the selected VHHs with PCV2 Cap protein.The white clones in lane A indicate that the bait (pBD-Cap) has no autoactivator activity in yeast. When DNA-BD/Cap was cotransformed into Y2HGold yeast strain with 12 random positive AD/VHH plasmids, all co-transformants appeared blue on both DDO/X/A and QDO/X/A plates (Figure 4, lanes B-M). In the control group (Figure 4, lane N), the pGBKT7-53 and pGADT7-T co-transformants (positive control) appeared blue on both DDO/X/A and QDO/X/A plates, whereas pGBKT7-Lam and pGADT7-T co-transformants (negative control) appeared white on the DDO/X/A plate and did not grow on the QDO/X/A plate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585807&req=5

pone-0056222-g004: Intracellular interaction of the selected VHHs with PCV2 Cap protein.The white clones in lane A indicate that the bait (pBD-Cap) has no autoactivator activity in yeast. When DNA-BD/Cap was cotransformed into Y2HGold yeast strain with 12 random positive AD/VHH plasmids, all co-transformants appeared blue on both DDO/X/A and QDO/X/A plates (Figure 4, lanes B-M). In the control group (Figure 4, lane N), the pGBKT7-53 and pGADT7-T co-transformants (positive control) appeared blue on both DDO/X/A and QDO/X/A plates, whereas pGBKT7-Lam and pGADT7-T co-transformants (negative control) appeared white on the DDO/X/A plate and did not grow on the QDO/X/A plate.
Mentions: Before Y2H experiments, we first confirmed that the BD-Cap fusion protein had no autoactivator activity in yeast (Figure 4, lane A). After mating the Y2HGold (pBD-Cap) and Y187 (pAD-VHHs) yeast libraries and incubating on DDO/X/A plates for 3–5 days, hundreds of yeast blue colonies were selected. However, only 36 clones were recovered after two more rounds of screening on QDO/X/A plates. The prey plasmids from the above 36 clones were isolated, recovered, and co-transformed with the bait plasmid (pBD-Cap) or pGBKT7 (empty insertion) to verify the positive interactions between VHHs and Cap protein in yeast strain Y2HGold. The co-transformation results showed that all 36 clones rescued from QDO/X/A plates were genuine positive clones. Lanes B-M of Figure 4 show 12 randomly selected positive clones. Meanwhile, both positive (pGBKT7-53 and pGADT7-T co-transformants) and negative (pGBKT7-Lam and pGADT7-T co-transformants) control groups were found eligible (Figure 4, lane N). The results for intracellular interaction between Cap and anti-Cap VHHs in yeast are also summarized in the Table S3 (Table S3 in File SI). Sequencing results showed that the 36 positive clones were of 21 different clonal origins and the insertions were homologous to the properties of camel VHH (Figure 5).

Bottom Line: Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences.Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells.All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, China.

ABSTRACT
Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs) are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H) library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×10(6) cfu/3 µg and 2×10(9) cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

Show MeSH
Related in: MedlinePlus