Agarose gel electrophoresis of <i>Camelus Bactrianus</i> VHH repertoire amplified by two successive PCRs.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056222#pone-0056222-g003" target="_blank">Figure 3A</a> as template. M in A and B indicate the DL2000 DNA marker. The primers used in two successive PCRs are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056222#pone-0056222-t001" target="_blank">Table 1</a>, and the schematics on the right of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056222#pone-0056222-g003" target="_blank">Figure 3A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056222#pone-0056222-g003" target="_blank">Figure 3B</a> represent the classical Abs (top), HCAbs (middle), and VHH (bottom).</p

Purification of positive VHHs and Cap proteins.

<p>Lanes A–I, The purification of VHHs was identified by 12% SDS-PAGE. VHHs were expressed by fusion of 6×His tag at N- terminus and purified on Talon Metal Affinity Resin (Clontech).The molecular weight of the 6×His-VHH fusion proteins were 16 kDa–18 kDa. Lane J and K, Purification of 70 kDa MBP-Cap fusion protein (42 kDa MBP tag plus 28 kDa Cap) and 42 kDa MBP tag on Amylose Resin (NEB), respectively. The two M’s represented the protein MW markers (sizes in kilodalton are indicated on the left side). On the right box, the letters represent the names of the corresponding proteins.</p

Determination of the specific interaction between Cap and anti-Cap VHHs by indirect ELISA.

<p>A, The plate was coated with PCV2 viral lysis (10^−6.4 TCID₅₀, 1∶800 dilution). B, The plate was coated with purified Cap protein (0.5 µg/ml). The positive serum in A and B was the anti-Cap serum taken from a pig. Negative 1 and 2 in A represent the wells coated with BSA (negative control 1) or the lysis of PCV2 uninfected FPRC cells (negative control 2), respectively. Negative 1 and in B represent the wells coated with BSA (negative control 1) and 0.5 µg/ml purified MBP tag (negative control 2). The absorbance at 450 nm indicated the binding activity between selected VHHs and Cap. Data is plotted as the average of 3 wells, with error bars representing the standard deviation.</p

Schematic of strategies for constructing a phage library and a Y2H library.

<p>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 <i>Camelus Bactrianus</i> VHH Y2H library construction are in the shaded box. The discontinuous arrow shows the schematic of the hallmark VHH domains.</p

Aligned amino acid sequence of 21 anti-Cap specific VHH antibody fragments.

<p>Amino acid sequences were aligned according to the Kabat numbering <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056222#pone.0056222-Harmsen1" target="_blank">[16]</a>. The dots denote the same sequences compared with VHH1. Differences in the sequence are shadowed, and the dash shows the missing sequences. The hallmark Cys residues are denoted by the thick-line boxes. The four conservative hallmark residues of VHH in FR2 (Val37Phe, Gly44Glu/Lys, Arg45Leu, and Trp47Gly) are denoted by the dotted line boxes.</p

Schematic of two alternate approaches for the selection of antigen-specific VHHs (continuation of <b>Figure 1</b>).

<p>On the left side, a VHH screening approach based on phage panning is presented. On the right side, a VHH screening approach based on Clontech Matchmaker™ Gold Yeast Two-Hybrid System is presented.</p

Primers used in this work.

<p>Primers used in this work.</p

Immunocytochemistry analysis with VHH3 mAb.

<p>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 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.</p

Design and Selection of a Camelid Single-Chain Antibody Yeast Two-Hybrid Library Produced <i>De Novo</i> for the Cap Protein of Porcine Circovirus Type 2 (PCV2)

<div><p>Nanobodies (or <u>v</u>ariable domain of the <u>h</u>eavy chain of the <u>h</u>eavy-chain antibodie<u>s</u>, 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 <i>Camelus Bactrianus</i> 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 7.26×10⁶ cfu/3 µg and 2×10⁹ 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 <i>in vivo</i> VHH throughput screening based on Y2H strategy.</p></div