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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

Agarose gel electrophoresis of Camelus Bactrianus VHH repertoire amplified by two successive PCRs.A, First round PCR to distinguish VH from VHH based on amplicon sizes. The upper bands in lanes 1–5 (∼900 bp) represent the Leader-VH-CH1-Hinge-CH2 region of classical Abs. The lower band (∼600 bp) in lanes 1–5 represents the Leader-VHH-Hinge-CH2 region of HCAbs. B, The complete VHH fragments is amplified (∼400 bp in lanes 1–2) by a second nested PCR using the purified 600 bp DNA from Figure 3A as template. M in A and B indicate the DL2000 DNA marker. The primers used in two successive PCRs are from Table 1, and the schematics on the right of Figure 3A and Figure 3B represent the classical Abs (top), HCAbs (middle), and VHH (bottom).
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pone-0056222-g003: Agarose gel electrophoresis of Camelus Bactrianus VHH repertoire amplified by two successive PCRs.A, First round PCR to distinguish VH from VHH based on amplicon sizes. The upper bands in lanes 1–5 (∼900 bp) represent the Leader-VH-CH1-Hinge-CH2 region of classical Abs. The lower band (∼600 bp) in lanes 1–5 represents the Leader-VHH-Hinge-CH2 region of HCAbs. B, The complete VHH fragments is amplified (∼400 bp in lanes 1–2) by a second nested PCR using the purified 600 bp DNA from Figure 3A as template. M in A and B indicate the DL2000 DNA marker. The primers used in two successive PCRs are from Table 1, and the schematics on the right of Figure 3A and Figure 3B represent the classical Abs (top), HCAbs (middle), and VHH (bottom).

Mentions: According to the procedure in Figure 1, two PCR products including a 900 bp fragment for VH-CH1-CH2 exons and 600 bp for VHH-CH2 exons, were amplified with primers CALL01 and CALL02 (Table 1 and Figure 3A). The only VHH domain (about 400 bp) was further amplified using the template of the 600 bp fragment isolated from Figure 3A as well as the primers VHH-up and VHH-down (Table 1 and Figure 3B).


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)

Agarose gel electrophoresis of Camelus Bactrianus VHH repertoire amplified by two successive PCRs.A, First round PCR to distinguish VH from VHH based on amplicon sizes. The upper bands in lanes 1–5 (∼900 bp) represent the Leader-VH-CH1-Hinge-CH2 region of classical Abs. The lower band (∼600 bp) in lanes 1–5 represents the Leader-VHH-Hinge-CH2 region of HCAbs. B, The complete VHH fragments is amplified (∼400 bp in lanes 1–2) by a second nested PCR using the purified 600 bp DNA from Figure 3A as template. M in A and B indicate the DL2000 DNA marker. The primers used in two successive PCRs are from Table 1, and the schematics on the right of Figure 3A and Figure 3B represent the classical Abs (top), HCAbs (middle), and VHH (bottom).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056222-g003: Agarose gel electrophoresis of Camelus Bactrianus VHH repertoire amplified by two successive PCRs.A, First round PCR to distinguish VH from VHH based on amplicon sizes. The upper bands in lanes 1–5 (∼900 bp) represent the Leader-VH-CH1-Hinge-CH2 region of classical Abs. The lower band (∼600 bp) in lanes 1–5 represents the Leader-VHH-Hinge-CH2 region of HCAbs. B, The complete VHH fragments is amplified (∼400 bp in lanes 1–2) by a second nested PCR using the purified 600 bp DNA from Figure 3A as template. M in A and B indicate the DL2000 DNA marker. The primers used in two successive PCRs are from Table 1, and the schematics on the right of Figure 3A and Figure 3B represent the classical Abs (top), HCAbs (middle), and VHH (bottom).
Mentions: According to the procedure in Figure 1, two PCR products including a 900 bp fragment for VH-CH1-CH2 exons and 600 bp for VHH-CH2 exons, were amplified with primers CALL01 and CALL02 (Table 1 and Figure 3A). The only VHH domain (about 400 bp) was further amplified using the template of the 600 bp fragment isolated from Figure 3A as well as the primers VHH-up and VHH-down (Table 1 and Figure 3B).

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