J Vet Sci.  2016 Mar;17(1):119-122. 10.4142/jvs.2016.17.1.119.

Molecular cloning, purification and immunogenicity of recombinant Brucella abortus 544 malate dehydrogenase protein

• Reyes AW
• Simborio H
• Hop HT
• Arayan LT
• Kim S

• KMID: 2363344
• DOI: http://doi.org/10.4142/jvs.2016.17.1.119

Abstract

The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis.

Keyword

Brucella; expression; immunogenicity; malate dehydrogenase; recombinant protein

MeSH Terms

Animals
Antigens, Bacterial/*immunology
Brucella abortus/*enzymology/immunology
Brucellosis/diagnosis/*veterinary
Cattle
Cattle Diseases/*diagnosis
Cloning, Molecular
Enzyme-Linked Immunosorbent Assay
Escherichia coli/genetics
Malate Dehydrogenase/*genetics/*immunology/isolation & purification
Mice
Recombinant Proteins/genetics/*immunology
Antigens, Bacterial
Malate Dehydrogenase
Recombinant Proteins

Figure

• 

  Fig. 1 Amplification of Brucella (B.) abortus mdh gene. PCR products on 1.5% (w/v) agarose gel. Lane 1, DNA marker; Lane 2, single expected band of mdh gene (963 bp, arrow).

• 

  Fig. 2 Expression and purification of B. abortus malate dehydrogenase (MDH) in pMAL expression system vector with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie brilliant blue staining. Lane 1, Protein Marker; Lane 2, total E. coli DH5α lysates; Lane 3, uninduced rMDH; Lane 4, induced unpurified rMDH; Lane 5, purified rMDH (approximately 76 kDa, arrow).

• 

  Fig. 3 Expression, purification and immunogenicity of B. abortus MDH in pMAL expression system vector. (A) SDS-PAGE profiles of rMDH (approximately 76 kDa) and empty pMAL vector (approximately 43 kDa) separated and stained with Coomassie brilliant blue. Lane M, protein marker; Lane 1, empty pMAL vector; Lane 2, purified rMDH. (B) Western blot analysis using Brucella-positive bovine serum at early (Lane 1, empty pMAL vector; Lane 2, purified rMDH), middle (Lane 3, empty pMAL vector; Lane 4, purified rMDH) and late stages of infection (Lane 5, empty pMAL vector; Lane 6, purified rMDH), and Brucella-negative bovine serum (Lane 7, empty pMAL vector; Lane 8, purified rMDH). (C) Western blot analysis using Brucella-positive mouse serum at early (Lane 1, empty pMAL vector; Lane 2, purified rMDH), middle (Lane 3, empty pMAL vector; Lane 4, purified rMDH) and late stages of infection (Lane 5, empty pMAL vector; Lane 6, purified rMDH), and Brucella-negative mouse serum (Lane 7, empty pMAL vector; Lane 8, purified rMDH).

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