FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / J Clin Microbiol / High-level expression and purification of a truncated merozo...
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.
Get Full Text
Publication Date: 2003-03-08 PubMed ID: 12624044PubMed Central: PMC150322DOI: 10.1128/JCM.41.3.1147-1151.2003Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • 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

                  References

                  This article includes 18 references
                  1. Avarzed A, De Waal DT, Igarashi I, Saito A, Oyamada T, Toyoda Y, Suzuki N. Prevalence of equine piroplasmosis in Central Mongolia.. Onderstepoort J Vet Res 1997 Jun;64(2):141-5.
                    pubmed: 9352563
                  2. Avarzed A, Igarashi I, De Waal DT, Kawai S, Oomori Y, Inoue N, Maki Y, Omata Y, Saito A, Nagasawa H, Toyoda Y, Suzuki N. Monoclonal antibody against Babesia equi: characterization and potential application of antigen for serodiagnosis.. J Clin Microbiol 1998 Jul;36(7):1835-9.
                    pmc: PMC104937pubmed: 9650921doi: 10.1128/jcm.36.7.1835-1839.1998google scholar: lookup
                  3. Brüning A. Equine piroplasmosis an update on diagnosis, treatment and prevention.. Br Vet J 1996 Mar;152(2):139-51.
                    pubmed: 8680838doi: 10.1016/s0007-1935(96)80070-4google scholar: lookup
                  4. Burg JL, Perelman D, Kasper LH, Ware PL, Boothroyd JC. Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii.. J Immunol 1988 Nov 15;141(10):3584-91.
                    pubmed: 3183382
                  5. Chen XG, Gong Y, Hua-Li, Lun ZR, Fung MC. High-level expression and purification of immunogenic recombinant SAG1 (P30) of Toxoplasma gondii in Escherichia coli.. Protein Expr Purif 2001 Oct;23(1):33-7.
                    pubmed: 11570843doi: 10.1006/prep.2001.1483google scholar: lookup
                  6. Holbrook AA. Biology of equine piroplasmosis.. J Am Vet Med Assoc 1969 Jul 15;155(2):453-4.
                    pubmed: 5816130
                  7. Hopp TP, Woods KR. Prediction of protein antigenic determinants from amino acid sequences.. Proc Natl Acad Sci U S A 1981 Jun;78(6):3824-8.
                    pmc: PMC319665pubmed: 6167991doi: 10.1073/pnas.78.6.3824google scholar: lookup
                  8. Jack RM, Ward PA. Mechanisms of entry of Plasmodium and Babesia into red cells. p. 445-458. In M. Ristic and J. P. Kreier (ed.), Babesiosis. Academic Press, Inc., New York, N.Y..
                  9. Knowles DP Jr, Perryman LE, Kappmeyer LS, Hennager SG. Detection of equine antibody to Babesia equi merozoite proteins by a monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay.. J Clin Microbiol 1991 Sep;29(9):2056-8.
                    pmc: PMC270260pubmed: 1774334doi: 10.1128/jcm.29.9.2056-2058.1991google scholar: lookup
                  10. Knowles DP, Kappmeyer LS, Perryman LE. Genetic and biochemical analysis of erythrocyte-stage surface antigens belonging to a family of highly conserved proteins of Babesia equi and Theileria species.. Mol Biochem Parasitol 1997 Dec 1;90(1):69-79.
                    pubmed: 9497033doi: 10.1016/s0166-6851(97)00150-3google scholar: lookup
                  11. Knowles RC. Equine babesiosis: epidemiology, control, and chemotherapy. Equine Vet. Sci. 8:61-64.
                  12. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.. Nature 1970 Aug 15;227(5259):680-5.
                    pubmed: 5432063doi: 10.1038/227680a0google scholar: lookup
                  13. McGuire TC, Van Hoosier GL Jr, Henson JB. The complement-fixation reaction in eguine infectious anemia: demonstration of inhibition by IgG (T).. J Immunol 1971 Dec;107(6):1738-44.
                    pubmed: 5000991
                  14. Tanaka T, Xuan X, Ikadai H, Igarashi I, Nagasawa H, Fujisaki K, Mikami T, Suzuki N. Expression of Babesia equi merozoite antigen-2 by recombinant baculovirus and its use in the ELISA.. Int J Parasitol 1999 Nov;29(11):1803-8.
                    pubmed: 10616926doi: 10.1016/s0020-7519(99)00146-0google scholar: lookup
                  15. Tenter AM, Friedhoff KT. Serodiagnosis of experimental and natural Babesia equi and B. caballi infections.. Vet Parasitol 1986 Mar;20(1-3):49-61.
                    pubmed: 3518217doi: 10.1016/0304-4017(86)90092-0google scholar: lookup
                  16. Weiland G. Species-specific serodiagnosis of equine piroplasma infections by means of complement fixation test (CFT), immunofluorescence (IIF), and enzyme-linked immunosorbent assay (ELISA).. Vet Parasitol 1986 Mar;20(1-3):43-8.
                    pubmed: 3518216doi: 10.1016/0304-4017(86)90091-9google scholar: lookup
                  17. Xuan X, Larsen A, Ikadai H, Tanaka T, Igarashi I, Nagasawa H, Fujisaki K, Toyoda Y, Suzuki N, Mikami T. Expression of Babesia equi merozoite antigen 1 in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.. J Clin Microbiol 2001 Feb;39(2):705-9.
                    pmc: PMC87800pubmed: 11158131doi: 10.1128/jcm.39.2.705-709.2001google scholar: lookup
                  18. Xuan X, Nagai A, Battsetseg B, Fukumoto S, Makala LH, Inoue N, Igarashi I, Mikami T, Fujisaki K. Diagnosis of equine piroplasmosis in Brazil by serodiagnostic methods with recombinant antigens.. J Vet Med Sci 2001 Oct;63(10):1159-60.
                    pubmed: 11714038doi: 10.1292/jvms.63.1159google scholar: lookup

                  Citations

                  This article has been cited 8 times.
                  1. Ceylan O, Xuan X, Sevinc F. Primary Tick-Borne Protozoan and Rickettsial Infections of Animals in Turkey.. Pathogens 2021 Feb 19;10(2).
                    doi: 10.3390/pathogens10020231pubmed: 33669573google scholar: lookup
                  2. Elata A, Mossaad E, Satti R, Matar N, Ohari Y, Xuan X, Inoue N, Suganuma K. Serological and molecular detection of selected hemoprotozoan parasites in donkeys in West Omdurman, Khartoum State, Sudan.. J Vet Med Sci 2020 Mar 5;82(3):286-293.
                    doi: 10.1292/jvms.19-0534pubmed: 31969541google scholar: lookup
                  3. Mans BJ, Pienaar R, Latif AA. A review of Theileria diagnostics and epidemiology.. Int J Parasitol Parasites Wildl 2015 Apr;4(1):104-18.
                    doi: 10.1016/j.ijppaw.2014.12.006pubmed: 25830110google scholar: lookup
                  4. Sevinc F, Cao S, Xuan X, Sevinc M, Ceylan O. Identification and expression of Babesia ovis secreted antigen 1 and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.. J Clin Microbiol 2015 May;53(5):1531-6.
                    doi: 10.1128/JCM.03219-14pubmed: 25694531google scholar: lookup
                  5. Wang M, Guo W, Igarashi I, Xuan X, Wang X, Xiang W, Jia H. Epidemiological investigation of equine piroplasmosis in China by enzyme-linked immunosorbent assays.. J Vet Med Sci 2014 Apr;76(4):549-52.
                    doi: 10.1292/jvms.13-0477pubmed: 24292247google scholar: lookup
                  6. Huang X, Xuan X, Verdida RA, Zhang S, Yokoyama N, Xu L, Igarashi I. Immunochromatographic test for simultaneous serodiagnosis of Babesia caballi and B. equi infections in horses.. Clin Vaccine Immunol 2006 May;13(5):553-5.
                    doi: 10.1128/CVI.13.5.553-555.2006pubmed: 16682475google scholar: lookup
                  7. Huang X, Xuan X, Xu L, Zhang S, Yokoyama N, Suzuki N, Igarashi I. Development of an immunochromatographic test with recombinant EMA-2 for the rapid detection of antibodies against Babesia equi in horses.. J Clin Microbiol 2004 Jan;42(1):359-61.
                    doi: 10.1128/JCM.42.1.359-361.2004pubmed: 14715778google scholar: lookup
                  8. Huang X, Xuan X, Hirata H, Yokoyama N, Xu L, Suzuki N, Igarashi I. Rapid immunochromatographic test using recombinant SAG2 for detection of antibodies against Toxoplasma gondii in cats.. J Clin Microbiol 2004 Jan;42(1):351-3.
                    doi: 10.1128/JCM.42.1.351-353.2004pubmed: 14715776google scholar: lookup
                  Subscribe to our newsletter
                  Join 99,357 horse owners like you who receive our email updates on horse health and nutrition.