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

JaeKeun Park; Chang-Il Suh; Nam Seok Lee; Sung-Geun Lee; Doo-Sung Cheon; Mi-Hwa Oh; Se Hwan Oh; Jeong Woong Park; Soon-Young Paik

doi:10.4167/jbv.2010.40.2.83

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Park, Suh, Lee, Lee, Cheon, Oh, Oh, Park, and Paik: Antibody Production of Baculovirus-expressed VP6 from Porcine Group C Rotavirus

Original Article

J Bacteriol Virol 2010;40(2):83-89.

Published online: 18 January 2010

DOI: https://doi.org/10.4167/jbv.2010.40.2.83

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

JaeKeun Park¹, Chang-Il Suh¹, Nam Seok Lee^1,², Sung-Geun Lee¹, Doo-Sung Cheon³, Mi-Hwa Oh⁴, Se Hwan Oh⁵, Jeong Woong Park⁶, Soon-Young Paik^1,^∗

¹Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul, Korea

²AdipoGen Inc., Incheon, Korea

³Division of Enteric and Hepatitis Viruses, Korea Centers for Disease Control and Prevention, Seoul, Korea

⁴National Institute of Animal Science, Rural Development Administration, Suwon, Korea

⁵Apgujung St. Mary's Eye Center, Seoul, Korea

⁶Senigen Co., Gwacheon, Korea

^∗Corresponding author: Soon-Young Paik. Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea. Phone: +82-2-2258-7342, Fax: +82-2-535-6473 e-mail: paik@catholic.ac.kr

^∗∗ This work was supported by the Korea Centers for Disease Control and Prevention project (2008-E00204-00) and Waterborne Virus Bank funded by National Research Foundation of Korea.

Revised 31 March 201030 April 2010 Accepted 12 May 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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™ 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.

Keywords: Porcine group C rotavirus, Baculovirus, VP6, Polyclonal antibody

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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)

Table 1.

Comparison of the nucleotide and deduced amino acid sequence homologies of the VP6 gene of CA-2 RVC with other rotavirus strains

Host species	Group	Strain	Accession numbers		Percent homology (%)
Host species	Group	Strain	Nucleotide	Amino acid	Nucleotide	Amino acid
Human	A	B1711	EF554086	ABU49770	51.6	40.4
Human	A	Hun5	EF554108	ABU49772	53.3	41.9
Bovine	A	WC3	AF411322	AAM73767	52.8	41.9
Avian	A	CH2	EF687020	ABV46497	50.2	42.2
Porcine	A	RU172	DQ204741	ABB17170	49.0	40.9
Human	B	WH-1	AY539858	AAT09116	37.6	15.7
Human	B	BANG373	AY238389	AAQ18653	37.6	15.9
Bovine	B	DB176	GQ358713	ADC53099	39.7	16.9
Human	C	Belem	M94155	AAA47339	82.6	90.7
Human	C	Bristol	X59843	CAA42504	82.8	90.7
Bovine	C	Shintoku	M88768	AAA03235	82.4	90.4
Porcine	C	Cowden	M94157	AAA47097	86.2	93.7

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