[Purification and reactivity of foot-and-mouth disease virus non-structural protein 3A, 3B and 2C expressed in E. coli]
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Published on: April 19, 2016
Yuanfang Fu1, Zengjun Lu, Yimei Cao
1State Key Laboratory of Veterinary Etiological Biology, National Foot-and-Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute of Chinese Academy of Agriculture Science, Lanzhou, China. wst258@163.com
Objective:
To purify and to detect reactivity of non-structural proteins 3A, 3B and 2C expressed in the Escherichia coli.
Methods:
FMDV NSP 3A, 3B and 2C containing the major B-cell antigenic sites were expressed in E. coli. We got renatured 2C protein by lysing of isolated inclusion body using high concentration of urea, and then diluted in a buffer system containing oxidized/reduced glutathione. Purified 3A, 3B and 2C were obtained by Ni-NTA His Bind Resin affinity chromatography. The reactivity of three NSPs with sera of different origin was measured using an indirect ELISA and Western-blot. The reactivity of three proteins was compared with 3ABC and 3D by detecting sera of clinically healthy sheep that were collected from epidemic region of Asia I FMD.
Results:
Proteins 3A and 3B were solubly expressed in bacteria, and 2C was expressed to form inclusion body. All three products could react specifically with sera from FMDV infected animal by western-blot and ELISA. The high coincident rates were observed between 3A, 3B, 2C and 3ABC.
Conclusion:
The results would provide useful materials for establishment of immunoelectro-transfer blot (EITB) diagnostic method, which could be used for differentiation of the FMDV infected animals from the vaccinated animals.
Insights
This study successfully purified Foot-and-Mouth Disease Virus (FMDV) non-structural proteins 3A, 3B, and 2C in E. coli. These proteins demonstrated specific reactivity, paving the way for improved diagnostic tools for FMDV.
Area of Science:
- Veterinary Virology
- Molecular Biology
- Immunology
Background:
- Foot-and-Mouth Disease Virus (FMDV) non-structural proteins (NSPs) are crucial targets for differentiating infected from vaccinated animals.
- Previous studies have focused on various FMDV NSPs, but efficient expression and reactivity assessment of 3A, 3B, and 2C remain important.
Purpose of the Study:
- To express and purify FMDV non-structural proteins 3A, 3B, and 2C in Escherichia coli.
- To evaluate the reactivity of these purified proteins with sera from FMDV-infected animals.
Main Methods:
- Proteins 3A, 3B, and 2C were expressed in E. coli.
- Purification involved Ni-NTA His Bind Resin affinity chromatography.
- Reactivity was assessed using indirect ELISA and Western-blotting with sera from infected animals and healthy sheep from an FMDV epidemic region.
Main Results:
- Proteins 3A and 3B were expressed solubly, while 2C formed inclusion bodies, which were renatured.
- All three purified proteins (3A, 3B, 2C) showed specific reactivity against FMDV-infected animal sera.
- High coincidence rates were observed between the reactivity of individual NSPs (3A, 3B, 2C) and the combined 3ABC antigen.
Conclusions:
- The purified FMDV NSPs 3A, 3B, and 2C are reactive and can be utilized as diagnostic reagents.
- These findings support the development of improved immunoelectro-transfer blot (EITB) assays for distinguishing FMDV-infected from vaccinated animals.
