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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

Immunocytochemistry analysis with VHH3 mAb.A and a, mock-infected FPRC cells; B and b, PCV2-infected FPRC cells. All cells were examined under an inverted light microscope. The expression of PCV2 Cap protein is visualized as a nigger-brown color in the nucleus, and cell nuclei stained with hematoxylin are shown in blue in the mock-infected FPRC cells. In PCV2-infection group, most of infected cells had already detached and lysed. So the cell coverage was much less than mock control.
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pone-0056222-g008: Immunocytochemistry analysis with VHH3 mAb.A and a, mock-infected FPRC cells; B and b, PCV2-infected FPRC cells. All cells were examined under an inverted light microscope. The expression of PCV2 Cap protein is visualized as a nigger-brown color in the nucleus, and cell nuclei stained with hematoxylin are shown in blue in the mock-infected FPRC cells. In PCV2-infection group, most of infected cells had already detached and lysed. So the cell coverage was much less than mock control.

Mentions: Using positive VHH3 clone as model, the intracellular specificity and binding affinity of selected VHHs were further detected by immunocytochemistry. As shown in Figure 8, in PCV2-infection group, most of infected cells had already detached and lysed. So the cell coverage was much less than mock control. Meanwhile, the strong nuclear brownish staining was specifically appeared in PCV2-infected FPRC cells group but not in the mock control (only FPRC cells). These results indicated the feasibility of detection intracellular PCV2 antigen with selected VHHs.


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)

Immunocytochemistry analysis with VHH3 mAb.A and a, mock-infected FPRC cells; B and b, PCV2-infected FPRC cells. All cells were examined under an inverted light microscope. The expression of PCV2 Cap protein is visualized as a nigger-brown color in the nucleus, and cell nuclei stained with hematoxylin are shown in blue in the mock-infected FPRC cells. In PCV2-infection group, most of infected cells had already detached and lysed. So the cell coverage was much less than mock control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056222-g008: Immunocytochemistry analysis with VHH3 mAb.A and a, mock-infected FPRC cells; B and b, PCV2-infected FPRC cells. All cells were examined under an inverted light microscope. The expression of PCV2 Cap protein is visualized as a nigger-brown color in the nucleus, and cell nuclei stained with hematoxylin are shown in blue in the mock-infected FPRC cells. In PCV2-infection group, most of infected cells had already detached and lysed. So the cell coverage was much less than mock control.
Mentions: Using positive VHH3 clone as model, the intracellular specificity and binding affinity of selected VHHs were further detected by immunocytochemistry. As shown in Figure 8, in PCV2-infection group, most of infected cells had already detached and lysed. So the cell coverage was much less than mock control. Meanwhile, the strong nuclear brownish staining was specifically appeared in PCV2-infected FPRC cells group but not in the mock control (only FPRC cells). These results indicated the feasibility of detection intracellular PCV2 antigen with selected VHHs.

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