Construction of a camelid VHH yeast two-hybrid library and the selection of VHH against haemagglutinin-neuraminidase protein of the Newcastle disease virus

Humoral immune response after immunization. Sera from IIama was collected, two-fold diluted and tested by HI using LaSota as antigen. Figure S1 Amplification of VHH through a nested PCR. (A) First round PCR to separate VH from VHH. The upper 900 bp bands represent the VH-CH1-Hinge-CH2 of conventional Abs (lane 1–8). The lower 600 bp bands represent the VHH-Hinge-CH2 of HCAbs (lane 1–8). (B) VHH amplified through nested PCR using 600 bp fragment recovered from first round PCR as template (lane 1–4). M in A and B was the DL2000 DNA marker. C in A and B represent the negative control. Figure S2 PCR identification of inserted VHH. 47 clones were randomly picked to determine the library functional diversity by PCR using universal primers T7 and 3’AD (Table 1). Meanwhile, Sterile water was used as negative controls. 45 clones have amplified the 500 bp VHH fragments (lane 1–47), while negative templates control haven’t amplified any bands (lane C). M indicated the DL2000 DNA marker. Figure S3 Detection of library capacity and library titer. (A) 10-3 dilution plating of the transformed cells calculated a library capacity of 1.25 × 107 independent clones. (B) 10-5 dilution plating of the cultured library indicated a library titer of 3.45 × 108 cfu/mL. Figure S4 Deduced amino acid aligment of 10 random picked VHH. Deduced amino acid sequences were analyzed according to the Kabat numbering. Differences in the sequences are pinked, and the dash represent the missing sequences. Two hallmark Cys residues are labeled by the thick-line boxes. The four conservative hallmark residues of VHH in FR2 are labeled by the dotted line boxes. Figure S5 pGBKT7-HN bait plasmid construction. (A) PCR was carried out to amplify a truncate HN gene (without transmembrane region) from La Sota strain. M, 5000 DNA marker. 1, Truncate HN. C, Negative control. (B) A truncate HN was cloned into pGBKT7 through BamH I and Sal I. M, 5000 DNA marker. 1, Double restriction enzyme digestion of pGBKT7-HN. Figure S6 pHSIE-VHH plasmid construction. (A) 7 positive VHH fragment were amplified from recovered positive clones containing pGADT7-VHH by PCR. M, 5000 DNA marker. 1–7, VHH 1–7. C, Negative control. (B) Double restriction enzyme digestion of pHSIE-VHHs. M, 5000 DNA marker. 1–7, pHSIE-VHH 1–7. Figure S7 Western blot analysis of bait protein expression. 2 mL of Y2HGold(pGBKT7-HN) culture liquid was extracted using yeast protein extraction reagent (Takara). c-Myc tag monoclonal antibody (1:4000 dilution) was used as first antibody and HRP-labeled goat anti-mouse antibody (1:5000) was used as second antibody. The immunoreactive was visualized with cECL Plus Western blotting detection reagent (CWBIO). (DOC 1129 kb

Newcastle disease virus V protein inhibits apoptosis in DF-1 cells by downregulating TXNL1

Abstract Many viral proteins are related to suppressing apoptosis in target cells and are hence beneficial to viral replication. The V protein of Newcastle disease virus (NDV) is one such protein that plays an important role in inhibiting apoptosis in a species-specific manner. However, to date, there have been no reports clarifying the antiapoptotic mechanisms of the V protein. The present study was undertaken to determine the apoptotic potential of the V protein in a chicken embryo fibroblast cell line (DF-1 cell) and to elucidate its molecular mechanisms of action. Here, a yeast two-hybrid system was used to screen the host proteins that interact with the V protein and identified thioredoxin-like protein 1 (TXNL1) as a potential binding partner. Immuno-colocalization of V protein and TXNL1 protein in DF-1 cells further verified the interaction of the two proteins. Through the overexpression of TXNL1 protein and knockdown of TXNL1 protein in DF-1 cells, the effects of NDV replication and cell apoptosis were examined. Cell apoptosis was detected by flow cytometry. The mRNA and protein expression levels of Bax, Bcl-2 and Caspase-3 were detected by quantitative real-time PCR (Q-PCR) and Western blotting. NDV expression was detected by Q-PCR and plaque assay. The results revealed that the TXNL1 protein induced apoptosis and inhibited NDV replication in DF-1 cells. Furthermore, the Western blot and Q-PCR results suggested that TXNL1 induced cell apoptosis through a pathway involving Bcl-2\Bax and Caspase-3. Finally, this work provides insight into the mechanism by which the V protein inhibits apoptosis

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

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

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

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