J Bacteriol Virol.  2010 Jun;40(2):83-89. 10.4167/jbv.2010.40.2.83.

Antibody Production of Baculovirus-expressed VP6 from Porcine Group C Rotavirus

• Park J
• Suh CI
• Lee NS
• Lee SG
• Cheon DS
• Oh MH
• Oh SH
• Park JW
• Paik SY

• KMID: 1456271
• DOI: http://doi.org/10.4167/jbv.2010.40.2.83

Abstract

The emerging pathogen, group C rotavirus (RVC) has been reported to cause acute diarrhea. But there was the limitation on the detection and monitoring for the absence of rapid sensitive diagnosis system. For the molecular biology study and diagnostic system development, we could detect porcine RVC by reverse transcriptase PCR (RT-PCR) analyses from 60 diarrheal disease porcine stool samples. VP6 full length RT-PCR product (CA-2 RVC, 1352 bp) was cloned and compared the nucleotide and deduced amino acid sequences with those of previously reported other porcine, human, and bovine rotavirus group A, B and C strains. Analyses data showed >82% homology on the nucleotide sequences and >90% homology on the deduced amino acid sequences with other RVCs. Recombinant baculovirus was prepared with cloned PCR product corresponding to VP6 coding sequence (CDS) (position 22~1206) into BaculoDirect(TM) C-term linear DNA, and used for the transfection of insect cells. The polyclonal antibody was produced from mice with purified recombinant VP6 and confirmed with western blot. Both of VP6 antigen and antibody, are useful for the development of rapid diagnostic system against RVC.

Keyword

Porcine group C rotavirus; Baculovirus; VP6; Polyclonal antibody

MeSH Terms

Amino Acid Sequence
Animals
Antibody Formation
Baculoviridae
Base Sequence
Blotting, Western
Clinical Coding
Clone Cells
Diarrhea
DNA
Humans
Insects
Mice
Molecular Biology
Polymerase Chain Reaction
Reverse Transcriptase Polymerase Chain Reaction
Rotavirus
Transfection
DNA

Figure

• 

  Figure 1. RT-PCR result for the detection of RVC with the 340 bp region of VP6 gene (A) and for the cloning of full length VP6 gene (B). (A) Lane 1, porcine RVC positive (340 bp); 2, negative control; M, 100 bp ladder. (B) Lane 1, full length VP6 (1352 bp); 2, negative control; M, 1 kb ladder.

• 

  Figure 2. Analysis of expression of recombinant VP6 protein from insect cells (A, B) and confirmation of activity of polyclonal antibody (C). (A) 12% SDS-PAGE analysis of recombinant VP6 protein (M, Protein size marker; Lane 1, 2 μg of cell lysate); (B) Western blot analysis of recombinant VP6 protein with anti-HisG antibody (1:5,000) (Lane 1, total cell lysate; 2, supernatant of cell lysate; 3, flowthrough; 4~5, washed; 6~8, eluted); (C) Western blot analysis using mouse polyclonal antibody (1:2,000) (Lane 1, 1.5 μg; 2, 3 μg of recombinant VP6 protein)

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