Phages harboring specific peptides that recognize the N protein of the porcine reproductive and respiratory syndrome virus distinguish the virus from other viruses

Abstract

The aim of the current study was to develop a novel diagnostic test for detecting porcine reproductive and respiratory syndrome virus (PRRSV) using phage display technology. The N gene of PRRSV isolate HH08 was cloned following reverse transcription-PCR. Sequence comparison indicated that the N gene shared 96.4% homology to that of North American PRRSV (isolate VR2332) and 35.5% with that of European PRRSV (isolate LV), indicating that the PRRSV isolate was related to the North American PRRSV genotype. The bacterially expressed N protein was used as a target in a biopanning process using a phage display random peptide library. Seven phages expressing different peptides had a specific binding activity with the N protein. The putative binding motifs were identified by DNA sequencing. More importantly, the selected phages harboring specific peptides that recognize the N protein of PRRSV were able to efficiently distinguish PRRSV from other viruses in enzyme-linked immunosorbent assays.

FIG. 1.

Schematic diagram of the PRRSV genome. The entire genome of PRRSV is provided. The leader sequence, poly(A) tail, RNA polymerase, and structural gene regions are indicated. The framed parts from the the N terminus (5′) to the C terminus (3′) are the identified open reading frames.

FIG. 2.

Schematic diagram of panning procedure. The panning steps of phage display technology are summarized. The “” symbol indicates the specific peptides on the ends of the phages; “” indicates target proteins; “▧” indicates immobilized support for a target. Other symbols on the ends of phages indicate unspecific exogenous peptides.

FIG. 3.

Phylogenetic relationships among the PRRSV isolates. Homologous identity among the PRRSVs is shown in panel a. The isolate names and isolate places are provided; sequence alignment at the amino acid level in the N region of the PRRSVs is shown in panel b. The majority sequence shown at top is the consensus sequence, and four amino acid changes between the NA prototype strain VR2332 and the HH08 isolate are boxed. Using the MEGALIGN program in DNASTAR with the Jotun Hein method, a constructed phylogenetic tree is shown in panel c. The isolate names, GenBank accession numbers, and isolation places are indicated.

FIG. 4.

Expression and purification of the PRRSV N protein. Protein expression was induced in the recombinant bacteria harboring the PRRSV N gene. SDS-PAGE results are provided. Lane 1, protein molecular mass marker; lane 2, control bacteria bearing the empty vector; lane 3, soluble N protein; lane 4, inclusion body N protein; lane 5, purified N protein.

FIG. 5.

Analysis of binding of selected phages to the PRRSV N protein using ELISA. Ten selected phages, named phages a to j, were incubated with the PRRSV N protein in ELISA plates to test their binding activities for the protein as described in Materials and Methods. The ratio between the optical density (OD) value of tested individual phage and the OD value of the control is shown on the y axis; the individual phage and the control phage complex from the phage library are shown on the x axis.

FIG. 6.

Phage-based ELISA for differentiating PRRSV from other pathogens. Seven phages harboring specific peptides recognizing the PRRSV N protein were identified from the 10 selected phages and were designated phages 1 to 7. Phages 1, 2, 4, 5, 6, and 7 are identical to the phages b, i, h, d, e, and g; phage 3 is identical to phages a, c, f, and j indicated in Fig. 3 and Table 2. They were incubated with the various pathogens in ELISA plates. The ratio between the OD value of individual phage and the OD value of the control is shown on the y axis. “TVV” is the abbreviation of the trivalent vaccine against swine fever, swine erysipelas, and Pasteurella multocida. The phage control is the phage complex from the phage library.