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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

Schematic of strategies for constructing a phage library and a Y2H library.On the left side, the conventional method of constructing a VHH phage library is presented. On the right side, the novel method of constructing a VHH Y2H library is presented. The differences in the procedure for Camelus Bactrianus VHH Y2H library construction are in the shaded box. The discontinuous arrow shows the schematic of the hallmark VHH domains.
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pone-0056222-g001: Schematic of strategies for constructing a phage library and a Y2H library.On the left side, the conventional method of constructing a VHH phage library is presented. On the right side, the novel method of constructing a VHH Y2H library is presented. The differences in the procedure for Camelus Bactrianus VHH Y2H library construction are in the shaded box. The discontinuous arrow shows the schematic of the hallmark VHH domains.

Mentions: In the present study, a novel method of generating a VHH library was reported because of the fast and direct selection of VHHs in yeast without any in vitro pre-selection. Compared with the conventional VHH phage library, the VHH Y2H library in this study had the following distinct features. First, the antigen gene was expressed in vivo by fusion with GAL4 DNA-binding domains (DNA-BD/bait), which avoided time-consuming antigen expression and purification in vitro. The yeast VHH library had a unique advantage for difficult-to-express proteins or proteins with complex post-translational modifications, or for the determination of VHHs targets [9]. Second, the repertoire cloning of VHH into the DNA-AD/prey plasmid was based on SMARTer™ and recombination technology (Clontech), which was more efficient than conventional restriction enzyme digestion and T4 ligase ligation [10]. Third, compared with the currently available golden standard (i.e., analysis of the crystal structure of a given antigen-antibody complex), this approach was more effective and realistic for the precise domain mapping of linear epitopes, confirmation of non-linear epitopes or conformational epitope sensors, and detection of secondary binding partners [11]. The general construction procedures of a VHH phage library and a Y2H library were shown in Figure 1, the screening procedures comparisons of the two libraries were shown in Figure 2 and Table S1 (Table S1 in File SI), and the advantages and disadvantages comparisons of the two libraries were shown in Table S2 (Table S2 in File SI).


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)

Schematic of strategies for constructing a phage library and a Y2H library.On the left side, the conventional method of constructing a VHH phage library is presented. On the right side, the novel method of constructing a VHH Y2H library is presented. The differences in the procedure for Camelus Bactrianus VHH Y2H library construction are in the shaded box. The discontinuous arrow shows the schematic of the hallmark VHH domains.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056222-g001: Schematic of strategies for constructing a phage library and a Y2H library.On the left side, the conventional method of constructing a VHH phage library is presented. On the right side, the novel method of constructing a VHH Y2H library is presented. The differences in the procedure for Camelus Bactrianus VHH Y2H library construction are in the shaded box. The discontinuous arrow shows the schematic of the hallmark VHH domains.
Mentions: In the present study, a novel method of generating a VHH library was reported because of the fast and direct selection of VHHs in yeast without any in vitro pre-selection. Compared with the conventional VHH phage library, the VHH Y2H library in this study had the following distinct features. First, the antigen gene was expressed in vivo by fusion with GAL4 DNA-binding domains (DNA-BD/bait), which avoided time-consuming antigen expression and purification in vitro. The yeast VHH library had a unique advantage for difficult-to-express proteins or proteins with complex post-translational modifications, or for the determination of VHHs targets [9]. Second, the repertoire cloning of VHH into the DNA-AD/prey plasmid was based on SMARTer™ and recombination technology (Clontech), which was more efficient than conventional restriction enzyme digestion and T4 ligase ligation [10]. Third, compared with the currently available golden standard (i.e., analysis of the crystal structure of a given antigen-antibody complex), this approach was more effective and realistic for the precise domain mapping of linear epitopes, confirmation of non-linear epitopes or conformational epitope sensors, and detection of secondary binding partners [11]. The general construction procedures of a VHH phage library and a Y2H library were shown in Figure 1, the screening procedures comparisons of the two libraries were shown in Figure 2 and Table S1 (Table S1 in File SI), and the advantages and disadvantages comparisons of the two libraries were shown in Table S2 (Table S2 in File SI).

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