Usefulness of Escherichia coli-expressed Recombinant VP6 Proteins of Group A Rotavirus in Serodiagosis of Rotavirus Infection

Ji-Hyun Seo; So-Young Kim; Ji Sook Park; Jae-Young Lim; Chan-Hoo Park; Hyang-Ok Woo; Hee-Shang Youn; Wonyong Kim; Hyung-Lyun Kang; Seung-Chul Baik; Woo-Kon Lee; Myung-Je Cho; Kwang-Ho Rhee

doi:10.5223/kjpgn.2010.13.2.134

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Seo, Kim, Park, Lim, Park, Woo, Youn, Kim, Kang, Baik, Lee, Cho, and Rhee: Usefulness of Escherichia coli-expressed Recombinant VP6 Proteins of Group A Rotavirus in Serodiagosis of Rotavirus Infection

Original Article

Gastrointestinal Tract

Korean Journal of Pediatric Gastroenterology and Nutrition

Korean Journal of Pediatric Gastroenterology and Nutrition 2010; 13(2): 134-145.

Published online: 30 September 2010

DOI: https://doi.org/10.5223/kjpgn.2010.13.2.134

Usefulness of Escherichia coli-expressed Recombinant VP6 Proteins of Group A Rotavirus in Serodiagosis of Rotavirus Infection

Ji-Hyun Seo, M.D., So-Young Kim, M.D., Ji Sook Park, M.D., Jae-Young Lim, M.D., Chan-Hoo Park, M.D., Hyang-Ok Woo, M.D., Hee-Shang Youn, M.D., Wonyong Kim, Ph.D.^†, Hyung-Lyun Kang, Ph.D.^*, Seung-Chul Baik, M.D.^*, Woo-Kon Lee, Ph.D.^*, Myung-Je Cho, Ph.D.^*, Kwang-Ho Rhee, M.D.^*

Department of Pediatrics, Gyeongsang National University School of Medicine, Institute of Health Science, Jinju, Korea.

^*Department of Microbiology, Gyeongsang National University School of Medicine, Institute of Health Science, Jinju, Korea.

^†Department of Microbiology, Chung-Ang University College of Medicine, Seoul, Korea.

Corresponding author (hsyoun@gnu.ac.kr)

Received 13 August 2010 Revised 17 August 2010 Accepted 3 September 2010

Abstract

Purpose

The serologic diagnosis of rotaviral infections is not commonly used in clinical practice, but is used in seroepidemiologic studies. In this study, the usefulness of Escherichia coli-expressed recombinant VP6 proteins of group A rotavirus in the serodiagnosis of rotavirus infections by ELISA was evaluated.

Methods

The recombinant VP6 proteins of group A rotavirus expressed in E. coli Rosetta II strain were purified and identified. One hundred sera from 22 children (4 healthy neonates, 13 healthy children, and 5 immunocompromised children) who had serial sera samples prior to and after rotavirus infections were provided by the Gyeongsang National University Hospital, a member of the National Biobank of Korea. IgG, IgA, and IgM antibodies against rVP6 were analyzed by ELISA in all of the patients and Western blot analysis in 4 neonates.

Results

ELISA tests using rVP6 proteins of group A rotavirus as antigen revealed that IgG, IgA, and IgM antibodies increased after rotaviral infections in most neonates and healthy children. IgG antibodies also increased after rotaviral infections in most immunocompromised children without an adequate increase in IgM or IgA antibodies. Western blot analysis in four neonates revealed very early IgM antibody responses, even in the sera with low optical densities in ELISA tests.

Conclusion

Our study showed that ELISA using rVP6 as an antigen is a valid diagnostic tool for seroepidemiologic studies of rotavirus infections and Western blot analysis is a sensitive test in detecting IgG, IgA, and and IgM antibodies in patients with rotavirus infections.

Keywords: Rotavirus, VP6, ELISA

Figures and Tables

Fig. 1

The nucleotide sequences of rotavirus VP6 gene was obtained from the National Center for Biotechnology Information (NCBI) GenBank.

Fig. 2

Amplicons with a size of 1,194 bp represent rotavirus VP6 (lanes 1~6).

Fig. 3

The recombinant proteins from recombinant E. coli pET15b/rotavirus VP6 were analyzed by electrophoresis (12% SDS-PAGE). M: protein size marker, 1: E. coli pET15b/rotavirus VP6 whole cell lysate without IPTG induction, 2: E. coli pET15b/rotavirus VP6 whole cell lysate with IPTG induction, 3: supernatant of the whole cell lysate with IPTG induction, 4: precipitate of the whole cell lysate with IPTG induction.

Fig. 4

The purified refolded rotavirus VP6 proteins were analyzed by electrophoresis (12% SDS-PAGE). M: protein size marker, 1: flow through, 2: wash out, 3~8: fractions eluted with 20, 40, 80, 160, 320, and 500 mM imidazole, respectively.

Fig. 5

Results of both ELISA and immunoblotting for detecting IgG, IgA, and IgM antibodies against rVP6 of rotavirus are shown in four healthy neonates who were diagnosed with rotaviral gastroenteritis. Sample date 0: the date when stool was confirmed positive for rotavirus antigen.

Fig. 6

Changes in optical densities of ELISA for detecting IgG, IgA, and IgM antibodies against rVP6 of rotavirus are shown in 13 healthy children who were diagnosed with rotaviral gastroenteritis. Sample date 0: the date when stool was confirmed positive for rotavirus antigen.

Fig. 7

Changes in optical densities of ELISA for detecting IgG, IgA, and IgM antibodies against rVP6 of rotavirus are shown in five immunocompromised children who were diagnosed with rotaviral gastroenteritis. Sample date 0: the date when stool was confirmed positive for rotavirus antigen.

Table 1

PCR Primer for Amplification of Rotavirus VP6 Gene

Table 2

Demographic Data and Clinical Characteristics of the Patients with Rotaviral Infections

^*He was treated with high dose intravenous gammaglobulin for acute ITP 15 days before rotaviral gastroenteritis. ITP: immune thrombocytopenic purpura, ALL: acute lymphoblastic leukemia, CML: chronic myelogenous leukemia.

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