Development of porcine rotavirus vp6 protein based ELISA for differentiation of this virus and other viruses

Abstract

Background: The context and purpose of the study included 1) bacterial expression of viral protein 6 (VP6) of porcine rotavirus (PRV) and generation of rabbit polyclonal antiserum to the VP6 protein; 3) establishment of a discrimination ELISA to distinguish PRV from a panel of other porcine viruses.

Results: The VP6 gene of PRV isolate DN30209 amplified by reverse transcription-PCR was 1356 bp containing a complete open reading frame (ORF) encoding 397 amino acids. Sequence comparison and phylogenetic analysis indicated that PRV DN30209 may belong to group A of rotavirus. Bacterially expressed VP6 was expressed in E.coli and anti-VP6 antibody was capable of distinguishing PRV from Porcine transmissible gastroenteritis virus, Porcine epidemic diarrhea virus, Porcine circovirus type II, Porcine reproductive and respiratory syndrome virus, Porcine pseudorabies virus and Porcine parvovirus.

Conclusions: PRV VP6 expressed in E. coli can be used to generate antibodies in rabbit; anti-VP6 serum antibody can be used as good diagnostic reagents for detection of PRV.

Figure 1

Homologous identity comparison of PRV VP6 gene. VP6 sequences of PRV isolates used in this study were subjected to multiple sequence alignments using the DNAStar software. Their grouping, name and origin are shown in Table 1.

Figure 2

RNA-Polyacrylamide gel electrophoresis (PAGE) of PRV. RNA-PAGE profile shows long electropherotype migration pattern of PRV DN30209 (lane 1). Lane 2 is negative sample.

Figure 3

VP6 amino acide-based phylogenetic tree. A phylogenetic tree was constructed based on these PRV VP6 amino acid sequences using DNASTAR software.

Figure 4

Bacterial expression of VP6 protein in E.coli. Expression of VP6 protein was detected by a Coomassie blue staining. PAGE gel screened with rabbit antiserum to the VP6 as the primary antibody. Lane 1, crude bacterial lysate from cells containing non-recombinant vector only; Lane 2, non-induced crude bacterial lysate from bacteria containing VP6 plasmid; Lane 3, protein marker; Lane 4 unlysed VP6-containing bacteria at 4 h post-induction; Lane 5, lysed VP6-containing bacterial supernatant at 4 h post-induction; Lane 6, lysed VP6-containing bacterial inclusion bodies. The size of protein marker and the VP6 protein is indicated.

Figure 5

Optimization of dilution of anti-VP6 antibody in ELISA. Purified VP6 protein (5 μg/well) was diluted and coated in ELISA wells, and the serially diluted anti-VP6 antibody was used as primary antibody in subsequent ELISA. Dilutions of anti-VP6 antibody are indicated. The OD₄₉₀ value of tested samples (P)/the OD₄₉₀ value of negative control, coating buffer (N)>2 is judged as positive and shown as a curve; the broken-line is trend line.

Figure 6

Western blot of VP6 protein. Empty vector-transforming bacterial protein (negative control) and VP6-bearing bacterial protein were transferred onto a nitrocellulose membrane, then, the membrane was consecutively incubated with the anti-VP6 antibody and HRP-conjugated secondary antibody. Their blot results are shown in lanes 2 and 3, respectively. Lane 1: protein marker. The size of protein marker and the VP6 protein is shown.

Figure 7

Immunofluorescence assay of cell surface expression of VP6. BHK cells were transfected with pVAX-VP6, then the cells were subjected to indirect immunofluorescence using anti-VP6 antiserum as primary antibody followed by incubation of FITC-conjugated secondary antibody (see M&M). A representative comparison is provided. A: vector-transfected cells with treatment of Triton X-100; B: vector-transfected cells without treatment of TritonX-100; C: VP6-transfected cells with treatment of Triton X-100; D: VP6-transfected cells without treatment of Triton X-100.

Figure 8

Discrimination ELISA. PRV, Porcine transmissible gastroenteritis virus (TGEV), Porcine epidemic diarrhea virus (PEDV), Porcine circovirus type II (PCV2), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine pseudorabies virus (PrV) and Porcine parvovirus (PPV), DMEM medium and virus lysis buffer were coated onto ELISA plates individually. The wells were incubated with the anti-PRV antibody followed by incubation of HRP-conjugated secondary antibody. The names of the pathogens are indicated in x axis; The OD₄₉₀ value of tested samples (P)/the OD₄₉₀ value of negative control, coating buffer (N)>2 is judged as positive. *compared with other group (p<0.01). The P/N values were the mean values from three independent assays.