Development of porcine rotavirus vp6 protein based ELISA for differentiation of this virus and other viruses
- Jiayi Zhu†1, 2,
- Qing Yang†3,
- Liyan Cao1,
- Xiujing Dou1,
- Jianguo Zhao4,
- Weijuan Zhu1,
- Fan Ding1, 5,
- Ri-e Bu2,
- Siqingaowa Suo1,
- Yudong Ren1,
- Guangxing Li1 and
- Xiaofeng Ren1, 2Email author
© Zhu et al.; licensee BioMed Central Ltd. 2013
Received: 23 February 2012
Accepted: 14 March 2013
Published: 22 March 2013
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.
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.
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.
KeywordsPRV VP6 gene Cloning Expression
Rotavirus, a member of family Reoviridae, is one of the causative agents of viral diarrhea in young children and animals worldwide [1–3]. Rotavirus is approximately 75 nm in diameter and has multilayered icosahedral protein capsid composed of an outer layer, an inner layer, and a core . The virus genome is comprised of 11 segments of double-stranded RNA, encoding six structural proteins (VP1-VP4, VP6, VP7) and six nonstructural proteins (NSP) [5, 6]. The outer capsid layer proteins of rotavirus, VP4 and VP7, may elicit neutralizing antibodies independently; in addition, the two proteins can be used to classify rotaviruses into P and G genotypes, respectively [7–9].
VP1, VP2 and VP3 proteins consist of the rotavirus core particles. VP1 was partially accessible to iodination in single-shelled particles . The accession degree of VP1 to the immune system may affect the reaction of anti-PRV to VP1 protein . VP2 is the most abundant structural protein in core particles . Anti-VP2 serum is a good indicator of prior infection due to its high immunogenicity . VP3 is a minor structural protein that may comigrate with the outer capsid protein VP4 in many gel systems and it may be involved in RNA replication; VP4 is cleaved to form VP5 and VP8 [4, 11].
VP6 is the major structural protein in virus particles located on the outer surface of single-shelled particles, in addition, VP6 is known as a subgroup antigen . VP6 is required not only for polymerase activity and but also for maintaining the proper structure of the viral core or the core protein-based transcriptional complex. Myristylation of VP6 is required for virus particle formation as well as targeting single-shelled particles to the ER membrane for budding . As VP6 is both highly immunogenic and antigenic, it is the most frequently used in diagnostic assays to detect PRV . Herein, we expressed the VP6 gene from a PRV Chinese isolate DN30209 by cloning this gene into a bacterial expression system. We then generated rabbit polyclonal anti-serum and ascertained its immunoreactivity to VP6 protein. Using the anti-VP6 antibody, we established a differentiating ELISA for PRV. These experiments provided basis for determination of PRV.
Results and discussion
Cloning and phylogeny of VP6 gene of PRV
Analysis of VP6 expression and immunoreactivities of its antibody
Discrimination ELISA for detection of PRV
Amplification of VP6 gene of PRV by PCR
PRV Chinese isolate named DN30209 was recently isolated in a pig farm in Heilongjiang Province, northeastern China. The virus was propagated in MA104 cells in presence of trypsin at a final concentration of 3 μg/mL (w/v). The viral total RNA was extracted with a commercial kit according to the manufacturer’s instructions (HaiGene, China). Sense primer (VP6-1): 5'- GGCTTTTAAACGAAGTCTTC and antisense primer (VP6-2): 5'- GGTCACATCCTCTCACTA were published primers  and used to amplify the VP6 gene for sequencing. The reverse transcription mixture included 5 μL of RNA (1 μg), 1 μL of primer VP6-2 (10 pmol), 4 μL of 5×RT-PCR buffer (TaKaRa, China),0.5 μL of RNase inhibitor, 1 μL of dNTP (10 mM/each), 1 μL of M-MLV reverse transcriptase (TaKaRa) and 7.5 μL of sterile water. The reaction was performed at 30°C for 10 min, 42°C for 60 min and 95°C for 5 min. Subsequent PCR mixture included the resulting 3 μL of cDNA (3 μg), 5 μL of 10×ExTaq buffer, 1 μL of each primer (10 pmol), 4 μL of dNTP (2.5 mM/each), 0.25 μL of ExTaqDNA (TaKaRa, China) and 35.75 μL of sterile water. The PCR was performed at 94°C for 5 min; 30 cycles of 94°C for 40 s, 52.8°C for 40 s, and 72°C for 1 min; a final extension at 72°C for 10 min. The PCR product was purified and subjected to DNA sequencing.
RNA-polyacrylamide gel electrophoresis (PAGE)
RNA of the PRV was extracted by mixing the virus suspension in PBS or PBS (negative control) with an equal volume of an extraction buffer (0.02 M Tris–HCl pH7.4, 0.3 M NaCl, 0.01 M MgCl2, 0.1% SDS, 5 μM EDTA, 4% sucrose and 0.04% bromophenol blue) followed by phenol–chloroform extraction at 12, 000 rpm and 4°C for 10 min. The resulting supernatant was subjected to RNA-PAGE to detect and analyze the genome of rotavirus. The RNA-PAGE was performed in 10% polyacrylamide slab gel using discontinuous buffer system without SDS as described by Laemmli  for 13 h at 10 mA. Viral RNA segments were visualized in silver staining.
Sequence comparison and phylogenetic analysis
Information of PRV VP6 genes used in this study
Procine rotavirus A
Procine rotavirus A
Procine rotavirus A
Procine rotavirus A
Procine rotavirus A
Procine rotavirus A
Procine rotavirus A
Rotavirus B stain
Procine rotavirus C
Human rotavirus C
Human rotavirus C
Human rotavirus B
Rotavirus C stain
Procine rotavirus A
Human rotavirus C
Procine rotavirus A
Construction of recombinant plasmids bearing VP6 gene and its bacterial expression
To construct expression plasmids bearing the VP6 gene, forward primer: 5'- CCCCGGATCCATGCAAAATTACGGA(VP6F) and reverse primer (VP6R) 5'- GGGGAAGCTTGTTAGACTCGGTAATA were self designed and used to re-amplify the VP6 gene using the obtained PCR product above as template. Both primers contained BamH I and EcoR I sites, respectively. Subsequent PCR was performed as above using the full-length VP6 gene as template. The PCR profile included 95°C for 5 min; 30 cycles of 95°C for 30 s, 52.8°C for 30 s, and 72°C for 2 min as well as a final extension at 72°C for 10 min. The resulting PCR product was inserted into the prokaryotic expression vector pET30a and eukaryotic expression vector pVAX-1 to generate recombinant plasmids pET30a-VP6 and pVAX-VP6, respectively.
The pET30a-VP6 was transformed into Escherichia coli (E. coli) Rosetta. The transformed E.coli was cultured in Luria-Bertani (LB) medium supplemented with Kanamycin (50 mg/mL) with shaking at 37°C. When OD600 reached 0.6, isopropyl beta-D-thiogalactoside (IPTG) was added to the medium to a final concentration of 1 mM to induce protein expression. Control culture containing the same bacteria transformed with empty vector was used as control. The expressing protein designated as Pro-VP6 was subjected to gel-purification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and renaturation by dialysis according to our previously reported protocols .
Generation of polyclonal antibody to VP6
Generation of polyclonal antiserum to Pro-VP6 was processed according to references with modifications [17–19, 28]. A New Zealand rabbit was immunized with 2 mL of gel-purified Pro-VP6 (1 mg/mL) emulsified with equal amounts of Freund’s complete adjuvant via subcutaneous injection. After ten days, 2 mL of the same antigen mixed with Freund’s incomplete adjuvant were injected into the rabbit weekly at two intervals. Antiserum was collected from the peripheral blood of the rabbit.
The titration of the antiserum and its reaction with VP6 protein was determined using ELISA: Purified VP6 was coated onto ELISA plates at a final concentration of 5 μg/well at 4°C overnight. The next day, the wells were incubated with blocking buffer at 37°C for 2 h, after three washes with PBS-0.1% Tween20 (PBST). The wells were incubated with serially diluted polyclonal antibodies at 37°C for 1 h. Then the plates were incubated with goat anti-rabbit IgG conjugated with peroxidase (1:5000 dilution in PBS) at 37°C for 1 h, after three washes with PBST. OPD (o-Phenylenediamine dihydrochloride) substrate (100 μL/well) was added and incubated for 15 min followed by stopping reaction with 2 M H2SO4. The OD490 values were determined an ELISA reader. In addition, the reactivity of the anti-VP6 antibody to PRV was also tested. Briefly, the purified PRV (104 PFU/mL) were coated onto ELISA plates (100 μL/well) and the dilution of antiserum to Pro-VP6 or the control serum was 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:210, 1:211 or 1:212. The other experimental steps were performed as above.
Pro-VP6-containing bacterial protein and empty vector-transforming bacterial protein (negative control) were subjected to SDS-PAGE and transferred to a nitrocellulose (NC) membrane. Rabbit antiserum to Pro-VP6 was diluted 1:2,000 in PBS and was used to incubate the membrane at room temperature for 1.5 h followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5000 dilution, Boster, China) for 1.5 h. The protein bands were visualized using OPD.
Indirect immunofluorescence assay (IFA)
Baby hamster kidney (BHK) cells seeded onto 24-well plates were transfected with pVAX-VP6 using polyethylenimine (PEI) transfection agent (Sigma, China) according to the manufacturer’s instructions. Empty vector-transfected cells were used as control. The cells were fixed with 4% PFA (polyformaldehyde) in PBS at room temperature for 20 min and quenched with 0.1 M glycine in PBS. Then, the cells were washed three times with PBST and treated with or without 1% Triton X-100 for 10 min followed by incubation with anti-VP6 serum (1:200 dilution in PBS containing 0.1% BSA) for 1 h. After three washes with PBST, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:200 in PBS containing 1% BSA) for 1 h at 37°C. After washing with PBST, the fluorescence signals were analyzed using fluorescence microscopy.
Immunoreactivity of the anti-VP6 antibody to other control viruses
To evaluate the immunoreactivity of the anti-VP6 antibody, a discrimination ELISA was established. PRV, Porcine transmissible gastroenteritis virus (TGEV) strain HR/DN1, Porcine epidemic diarrhea virus (PEDV) strain HLJBY, Porcine circovirus type II (PCV2) strain PCV2-LJR, Porcine reproductive and respiratory syndrome virus (PRRSV) strain JilinTN1, Porcine pseudorabies virus (PrV) strain Kaplan and Porcine parvovirus (PPV) strain PPV2010 were treated with lysis buffer (0.1% SDS, 10 mmol/liter Tris–HCl [pH 7.4], and 1 mmol/liter EDTA) followed by dilution in carbonate–bicarbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and the pathogens were coated into ELISA plates (8 μg/well) at 4°C overnight. Wells coated with DMEM or lysis buffer were used as control. The anti-PRV antibody was used as primary antibody in the above-mentioned ELISA to detect the pathogens. The OD490 value of tested samples (P)/the OD490 value of negative control, coating buffer (N)>2 was judged as positive. The experiment was performed in triplicate. Statistical analysis of the data was processed with SPSS 11.5 software; p<0.05 and p<0.01 were defined as statistically significant and statistically very significant, respectively.
Grant Sponsored by Chang Jiang Scholar Candidates Programme for Provincial Universities in Heilongjiang,CSCP, Program for New Century Excellent Talents in University of Ministry of Education of P.R. China (NCET-10-0144), and Research Team Program on Scientific and Technological Innovation in Heilongjiang Provincial University (2011TD001) to X.R.; Research Fund for the Doctoral Program of Higher Education of China (20114320120006), Major Research Foundation of Education Bureau of Hunan Province (11A050) to Q.Y.; National Natural Science Foundation of China (31172295) to G.L.; Supported by The State Key Laboratory of Reproductive Biology (Grant No. Y252694C01) to J.Z.; National Natural Science Foundation of China (31270187) to Y.R. are acknowledged for their support on Research.
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