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Journal of clinical microbiology2003; 41(3); 1147-1151; doi: 10.1128/JCM.41.3.1147-1151.2003

High-level expression and purification of a truncated merozoite antigen-2 of Babesia equi in Escherichia coli and its potential for immunodiagnosis.

Abstract: The gene encoding a truncated merozoite antigen-2 (EMA-2t) of Babesia equi was cloned and highly expressed in Escherichia coli as a glutathione S-transferase fusion protein (G-rEMA-2t). Both G-rEMA-2t and rEMA-2t (after the removal of glutathione S-transferase) had good antigenicity. Either Western blot analysis with rEMA-2t or enzyme-linked immunosorbent assay (ELISA) with G-rEMA-2t clearly discriminated the sera of horses experimentally infected with B. equi from sera of horses infected with Babesia caballi and healthy horses, although rEMA-2t was not suitable for ELISA, probably owing to its poor absorbability to the plates. The specific antibodies in B. equi-infected horses were detectable during both acute and latent infection (6 to 244 days postinfection). Horse sera from Jilin Province, China, were examined by the two tests. The seroprevalence of B. equi was 49.2% (31 of 63 sera) by Western blot analysis with rEMA-2t and 47.6% (30 of 63 sera) by ELISA with G-rEMA-2t. The correspondence was 98.4% (62 of 63 sera) between the two tests. The results indicate that G-rEMA-2t and rEMA-2t proteins should be suitable antigens for the development of an effective immunodiagnostic assay due to their high sensitivity, specificity, and great yield.
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Publication Date: 2003-03-08 PubMed ID: 12624044PubMed Central: PMC150322DOI: 10.1128/JCM.41.3.1147-1151.2003Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study presents the development of a diagnostic tool for the detection of Babesia equi, a parasitic disease in horses, by genetically engineering a bacteria, E. coli, to produce two versions of a key antigen (a substance that induces an immune response). The researchers found a high correlation of positive results between their new tests and confirmed cases of the disease.

Research Methodology

  • The research team chose the gene for EMA-2t, a variant of the merozoite antigen-2 from the Babesia equi parasite.
  • They introduced this gene into the bacterium Escherichia coli, causing the bacteria to produce the antigen as a fusion protein (a protein created from two or more genes that originally coded for separate proteins) with glutathione S-transferase. This fusion protein was named G-rEMA-2t.
  • The fusion protein was then purified, and the glutathione S-transferase was removed to create another protein called rEMA-2t.
  • Both proteins displayed good antigenicity, meaning they were able to provoke an immune response.

Testing and Results

  • The researchers conducted various tests on horse serum samples. They used rEMA-2t in a Western blot analysis, a method commonly used in molecular biology to detect specific protein molecules in a sample. ELISA, a test that uses antibodies and color change to identify a substance, was used with G-rEMA-2t.
  • These tests clearly distinguished between serum of horses infected with Babesia equi and those infected with a different species, Babesia caballi, as well as healthy horses. However, rEMA-2t was not appropriate for the ELISA test, most likely due to its weak absorbability to the plates used in the test.
  • Horse sera from Jilin Province, China, were also tested. The seroprevalence (the level of a pathogen in a population, measured in serum) of B. equi was found to be 49.2% using Western blot analysis with rEMA-2t and 47.6% using ELISA with G-rEMA-2t. There was near perfect agreement (98.4%) between the two tests.

Conclusion

  • The researchers concluded that these manufactured proteins could be effective antigens for successfully diagnosing Babesia equi due to their high sensitivity, specificity, and yield.
  • This proposed method could greatly enhance the early detection and control of Babesia equi, which in turn would help to improve the health and welfare of horses worldwide.

Cite This Article

APA
Huang X, Xuan X, Yokoyama N, Xu L, Suzuki H, Sugimoto C, Nagasawa H, Fujisaki K, Igarashi I. (2003). High-level expression and purification of a truncated merozoite antigen-2 of Babesia equi in Escherichia coli and its potential for immunodiagnosis. J Clin Microbiol, 41(3), 1147-1151. https://doi.org/10.1128/JCM.41.3.1147-1151.2003

Publication

Journal of clinical microbiology
ISSN: 0095-1137
NlmUniqueID: 7505564
Country: United States
Language: English
Volume: 41
Issue: 3
Pages: 1147-1151

Researcher Affiliations

Huang, Xiaohong
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
Xuan, Xuenan
    Yokoyama, Naoaki
      Xu, Longshan
        Suzuki, Hiroshi
          Sugimoto, Chihiro
            Nagasawa, Hideyuki
              Fujisaki, Kozo
                Igarashi, Ikuo

                  MeSH Terms

                  • Animals
                  • Antibody Specificity
                  • Antigens, Protozoan / biosynthesis
                  • Antigens, Protozoan / genetics
                  • Antigens, Protozoan / isolation & purification
                  • Babesia / chemistry
                  • Babesia / immunology
                  • Babesia / isolation & purification
                  • Babesiosis / diagnosis
                  • Babesiosis / immunology
                  • Blotting, Western
                  • China
                  • Cloning, Molecular
                  • Enzyme-Linked Immunosorbent Assay
                  • Escherichia coli / genetics
                  • Gene Deletion
                  • Horses
                  • Immunologic Tests
                  • Kinetics
                  • Peptide Fragments / biosynthesis
                  • Peptide Fragments / genetics
                  • Peptide Fragments / isolation & purification
                  • Protozoan Proteins / biosynthesis
                  • Protozoan Proteins / genetics
                  • Protozoan Proteins / isolation & purification
                  • Recombinant Proteins / biosynthesis
                  • Recombinant Proteins / genetics
                  • Recombinant Proteins / isolation & purification

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                  Citations

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