J Bacteriol Virol.  2011 Sep;41(3):173-181. 10.4167/jbv.2011.41.3.173.

Sequence Analysis and Expression of ORF5 in Korean Isolate of Porcine Reproductive and Respiratory Syndrome Virus

• Yi SS
• Sun JS
• Kim HS
• Kang SY
• Hahn TW

• KMID: 1449892
• DOI: http://doi.org/10.4167/jbv.2011.41.3.173

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of reproductive failure and respiratory disorders in pigs. The viral genome consists of eight overlapping open reading frames (ORFs). ORF5 encodes one of the major glycoproteins and is known as an immunologically important structural protein associated with virus neutralization. The ORF5 gene of the Korean PRRSV isolate, CNV-1, was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and sequenced. The nucleotide and amino acid sequences of CNV-1 ORF5 shared 91% and 83% identity, respectively, with the American isolate (VR2332 strain) and 57% and 49% identity with the European isolate. For the expression and easy purification of ORF5, the cDNA containing the complete ORF5 sequence fused in-frame with sequence encoding glutathione S-transferase (GST) was cloned into a baculovirus transfer vector and transfected into Sf9 cells. The GST-ORF5 fusion protein produced in Sf9 cells was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Sequencing results confirmed that the recombinant baculovirus from Sf9 cells contains the complete ORF5 gene. Further studies in this direction will address whether ORF5 can be a good candidate for a subunit vaccine against PRRSV in Korea.

Keyword

Porcine reproductive and respiratory syndrome virus (PRRSV); CNV-1; Open reading frame 5 (ORF5); Recombinant baculovirus

MeSH Terms

Amino Acid Sequence
Baculoviridae
Blotting, Western
Clone Cells
DNA, Complementary
Electrophoresis
Genome, Viral
Glutathione Transferase
Glycoproteins
Korea
Open Reading Frames
Porcine Reproductive and Respiratory Syndrome
Porcine respiratory and reproductive syndrome virus
Sequence Analysis
Sf9 Cells
Sodium
Swine
Viruses
DNA, Complementary
Glutathione Transferase
Glycoproteins
Sodium

Figure

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  Figure 1. Amplification of CNV-1 ORF5. Lanes: 1, 100 bp ladder; 2, CNV-1. The numbers on both sides indicate the size in bp.

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  Figure 2. Nucleotide sequences (A) and amino acid sequences (B) of CNV-1 ORF5 compared with those of VR2332 (accession number U87392) and LV (accession number NC_002533). Underlines indicate putative N-glycosylatio sites.

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  Figure 3. Analysis of protein expression by Western blottings probed with swine anti-PRRSV serum (A) and mouse anti-GST monospecific antibody (B). The supernatants and the cell pellet were harvested at four days post-infection and were separated by SDS-PAGE, then transferred to nitrocellulose membranes. The proteins were detected with peroxidase-labeled anti-pig IgG (A) and peroxidase-labeled anti-mouse IgG (B). Lanes: 1, lysate of Sf9 cells infected with recombinant baculovirus containing ORF5; 2, lysate of Sf9 cells infected with wild-type AcNPV; 3, lysate of Sf9 cells infected with recombinant Xy1E; 4, supernatant of Sf9 cells infected with recombinant baculovirus containing ORF5; 5, supernatant of Sf9 cells infected with wild-type AcNPV; and 6, lysate of non-infected Sf9 cells. The numbers at the left indicate size markers.

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