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Assembly of cholera toxin B subunit full-length rotavirus NSP4 fusion protein oligomers in transgenic potato

  • Genetic Transformation and Hybridization
  • Published:
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Abstract

A CTB-NSP4175 fusion gene encoding the entire 175-aa murine rotavirus NSP4 enterotoxin protein was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB-NSP4175 enterotoxin fusion gene was detected in the genomic DNA of transformed leaves by PCR DNA amplification. Synthesis and assembly of the full-length CTB-NSP4175 fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-NSP4175 fusion protein pentamers to intestinal epithelial cell membrane receptors was quantified by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). The ELISA results showed that CTB-NSP4175 fusion protein was 0.006–0.026% of the total soluble tuber protein. The synthesis of CTB-NSP4175 monomers and their assembly into biologically active oligomers in transformed potato tubers demonstrates the feasibility of using edible plants for the synthesis of enterocyte-targeted full-length rotavirus enterotoxin antigens that retain all of their pathogenic epitopes for initiation of a maximum mucosal immune response.

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Fig. 3 A, B.
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Abbreviations

2,4-D :

2,4-Dichlorophenoxyacetic acid

BA :

Benzyl adenine

BSA :

Bovine serum albumin

CT :

Cholera toxin

CTB :

Cholera toxin B subunit

CTBh :

CTB hinge

ELISA :

Enzyme-linked immunosorbent assay

ER:

Endoplasmic reticulum

IAA :

Indole-3-acetic acid

IFN-γ :

Interferon-γ

IL-2 :

Interleukin-2

LLU :

Loma Linda University

MS :

Murashige and Skoog basal medium

NAA :

Naphthaleneacetic acid

NSP4 :

Rotavirus enterotoxin non-structural protein

PBS :

Phosphate-buffered saline

PBST :

Phosphate-buffered saline containing 0.05% Tween-20

RLU :

Relative light unit

TSP :

Total soluble plant protein

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Acknowledgements

The authors would like to thank Dr. John Mekalanos, Harvard University Medical School, for the gift of plasmid pRT42 containing ctxAB from the Classical Vibrio cholerae strain 569B, and Dr. Mary Estes, Baylor College of Medicine, for plasmid pCR2.1-NSP4 containing the simian virus SA-11 gene 10 encoding the full-length NSP4175 protein. The authors would also like to thank the Korea Science and Engineering Foundation (KOSEF) for providing a postdoctoral fellowship to Dr. Kim that enabled him to contribute to this project. The research reported in this manuscript was supported by a subcontract from the National Medical Testbed (DAMD17-97-2-7016) to W.H.R.L and an intramural grant from Loma Linda University to W.H.R.L. The views opinions and/or findings contained in this report are those of the authors and should not be construed as a position, policy, decision or endorsement of the National Medical Technology Testbed Inc.

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  1. Department of Biochemistry and Microbiology and Center for Molecular Biology and Gene Therapy, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA

    T.-G. Kim & W. H. R. Langridge

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  1. T.-G. Kim
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  2. W. H. R. Langridge
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Correspondence to W. H. R. Langridge.

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Communicated by W.A. Parrott

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Kim, TG., Langridge, W.H.R. Assembly of cholera toxin B subunit full-length rotavirus NSP4 fusion protein oligomers in transgenic potato. Plant Cell Rep 21, 884–890 (2003). https://doi.org/10.1007/s00299-003-0599-4

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  • DOI: https://doi.org/10.1007/s00299-003-0599-4

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