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Expression of foot and mouth disease virus nonstructural polyprotein 3ABC with inactive 3C(pro) in Escherichia coli

Ladawan Sariya1, Nattarat Thangthumniyom, Worawidh Wajjwalku

  • 1Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.

Protein Expression and Purification
|August 4, 2011
PubMed

Insights

A mutated Foot-and-Mouth Disease Virus (FMDV) 3ABC protein, with inhibited protease activity, was successfully expressed in E. coli. This mutated 3ABC protein can differentiate between naturally infected and vaccinated animals, aiding diagnostic test development.

Area of Science:

  • Molecular Biology
  • Virology
  • Immunology

Background:

  • The nonstructural 3ABC protein of Foot-and-Mouth Disease Virus (FMDV) is crucial for differentiating between naturally infected and vaccinated animals.
  • FMDV 3ABC is a polyprotein processed by its own 3C(pro) protease into 3A, 3B, and 3C(pro) components.

Purpose of the Study:

  • To express and characterize a mutated 3ABC (mu3ABC) protein of FMDV with inhibited protease activity.
  • To evaluate the potential of the expressed mu3ABC protein in developing diagnostic tests for FMDV.

Main Methods:

  • Cloning and expression of native and mutated 3ABC genes in Escherichia coli.
  • Site-directed mutagenesis was used to abolish the protease activity of the 3C(pro) component in mu3ABC.
  • SDS-PAGE and Western blot analysis were employed to confirm protein expression and integrity.
  • Immunological assays were performed using sera from naturally infected and vaccinated cattle.

Main Results:

  • The mutated 3ABC (mu3ABC) protein was successfully expressed in E. coli, primarily in inclusion bodies, and detected as monomer and dimer.
  • Expression of full-length 3ABC was only achieved when the protease activity was abolished.
  • The mu3ABC protein showed strong reactivity with serum from naturally infected cattle but weak reactivity with serum from vaccinated cattle.

Conclusions:

  • Complete abolition of 3C(pro) protease activity is essential for the successful expression of full-length FMDV 3ABC protein.
  • The mu3ABC protein holds significant promise for the development of a reliable diagnostic test to distinguish FMDV-infected from vaccinated animals.

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