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Home / Equine Research Bank / J Clin Microbiol / Cloning and expression of a 48-kilodalton Babesia caballi me...
Journal of clinical microbiology1999; 37(11); 3475-3480; doi: 10.1128/JCM.37.11.3475-3480.1999

Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential use of the recombinant antigen in an enzyme-linked immunosorbent assay.

Abstract: A cDNA expression library prepared from Babesia caballi merozoite mRNA was screened with a monoclonal antibody BC11D against the rhoptry protein of B. caballi merozoite. A cDNA encoding a 48-kDa protein of B. caballi was cloned and designated BC48. The complete nucleotide sequence of the BC48 gene had 1,828 bp and was shown to contain no intron. Southern blotting analysis indicated that the BC48 gene contained more than two copies in the B. caballi genome. Computer analysis suggested that this sequence contained an open reading frame of 1,374 bp with a coding capacity of approximately 52 kDa. The recombinant protein expressed by the vaccinia virus vector in horse cells had an apparent molecular mass of 48 kDa, which was the same as that of the native B. caballi 48-kDa protein. Moreover, recombinant proteins expressed by the pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion proteins were used for antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate very clearly between B. caballi-infected horse sera and B. equi-infected horse sera or noninfected normal horse sera. These results suggest that this simple and highly sensitive test might be applicable to the detection of B. caballi-infected horses in the field.
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Publication Date: 1999-10-19 PubMed ID: 10523537PubMed Central: PMC85671DOI: 10.1128/JCM.37.11.3475-3480.1999Google Scholar: Lookup
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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.

The research paper describes the successful cloning and expression of a specific protein found in a parasite called Babesia caballi, and suggests the potential use of this recombinant protein as an antigen in a diagnostic test for the infection in horses.

Cloning and Expression of the BC48 Protein

  • The research team utilized a monoclonal antibody, known as BC11D, to screen a cDNA expression library that was prepared from Babesia caballi merozoite mRNA. This merozoite stage of the parasite is when it invades red blood cells, making it crucial to the survival of the parasite.
  • A cDNA encoding a 48-kilodalton protein of B. caballi was cloned and was assigned the name BC48. The entire nucleotide sequence of the BC48 gene had 1,828 base pairs (bp) and surprisingly contained no intron – a typically common non-coding section found within genes.
  • Southern blotting, a common molecular biology technique, indicated that the BC48 gene contained more than two copies within the B. caballi genome.

Characterization and Use of the Recombinant Protein

  • Computer analysis revealed that the BC48 sequence contained an open reading frame (the part of a gene that is possible to translate) of 1,374 bp, which theoretically would have a coding capacity of approximately 52 kilodaltons.
  • The recombinant BC48 protein was expressed using a vaccinia virus vector in horse cells, and parametrically demonstrated a molecular mass of 48 kilodaltons, identical to that of the original B. caballi 48-kDa protein.
  • Furthermore, the recombinant proteins were produced as fused proteins using a pGEX4T expression vector in E. coli. These proteins then acted as the antigens in an enzyme-linked immunosorbent assay (ELISA), a commonly used diagnostic tool in medicine and biology.

Development of a New Diagnostic Tool

  • The team discovered that the ELISA using the BC48 antigen was capable of clearly distinguishing between B. caballi-infected horse sera and B. equi-infected horse sera or uninfected horse sera.
  • Based on these findings, the team suggest that this new, simple, and highly sensitive test might be useful in the detection of B. caballi-infected horses in real-world scenarios, offering an improved method for diagnosing this parasitic infection.

Cite This Article

APA
Ikadai H, Xuan X, Igarashi I, Tanaka S, Kanemaru T, Nagasawa H, Fujisaki K, Suzuki N, Mikami T. (1999). Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential use of the recombinant antigen in an enzyme-linked immunosorbent assay. J Clin Microbiol, 37(11), 3475-3480. https://doi.org/10.1128/JCM.37.11.3475-3480.1999

Publication

Journal of clinical microbiology
ISSN: 0095-1137
NlmUniqueID: 7505564
Country: United States
Language: English
Volume: 37
Issue: 11
Pages: 3475-3480

Researcher Affiliations

Ikadai, H
  • The Research Center for Protozoan Molecular Immunology, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
Xuan, X
    Igarashi, I
      Tanaka, S
        Kanemaru, T
          Nagasawa, H
            Fujisaki, K
              Suzuki, N
                Mikami, T

                  MeSH Terms

                  • Amino Acid Sequence
                  • Animals
                  • Antigens, Protozoan / genetics
                  • Babesia / genetics
                  • Babesia / immunology
                  • Babesia / ultrastructure
                  • Babesiosis / diagnosis
                  • Base Sequence
                  • Cloning, Molecular
                  • DNA Primers / genetics
                  • DNA, Protozoan / genetics
                  • Enzyme-Linked Immunosorbent Assay / methods
                  • Escherichia coli / genetics
                  • Gene Expression
                  • Genes, Protozoan
                  • Horse Diseases / diagnosis
                  • Horses
                  • Microscopy, Immunoelectron
                  • Molecular Sequence Data
                  • Protozoan Proteins / genetics
                  • Protozoan Proteins / immunology
                  • Recombinant Fusion Proteins / genetics
                  • Recombinant Fusion Proteins / immunology

                  References

                  This article includes 30 references
                  1. Avarzed A, Igarashi I, Kanemaru T, Hirumi K, Omata Y, Saito A, Oyamada T, Nagasawa H, Toyoda Y, Suzuki N. Improved in vitro cultivation of Babesia caballi.. J Vet Med Sci 1997 Jun;59(6):479-81.
                    pubmed: 9234227doi: 10.1292/jvms.59.479google scholar: lookup
                  2. Böse R, Daemen K. Demonstration of the humoral immune response of horses to Babesia caballi by western blotting.. Int J Parasitol 1992 Aug;22(5):627-30.
                    pubmed: 1399247doi: 10.1016/0020-7519(92)90011-9google scholar: lookup
                  3. Böse R, Peymann B. Diagnosis of Babesia caballi infections in horses by enzyme-linked immunosorbent assay (ELISA) and western blot.. Int J Parasitol 1994 May;24(3):347-6.
                    pubmed: 8070952
                  4. Böse R, Peymann B, Barbosa IP. Identification of diagnostic antigens for South American Babesia caballi infections.. Int J Parasitol 1994 Apr;24(2):255-8.
                    pmc: PMC7130368pubmed: 8026903doi: 10.1016/0020-7519(94)90034-5google scholar: lookup
                  5. Brüning A, Phipps P, Posnett E, Canning EU. Monoclonal antibodies against Babesia caballi and Babesia equi and their application in serodiagnosis.. Vet Parasitol 1997 Jan;68(1-2):11-26.
                    pubmed: 9066047doi: 10.1016/s0304-4017(96)01074-6google scholar: lookup
                  6. Dalrymple BP, Casu RE, Peters JM, Dimmock CM, Gale KR, Boese R, Wright IG. Characterisation of a family of multi-copy genes encoding rhoptry protein homologues in Babesia bovis, Babesia ovis and Babesia canis.. Mol Biochem Parasitol 1993 Feb;57(2):181-92.
                    pubmed: 8433711doi: 10.1016/0166-6851(93)90194-3google scholar: lookup
                  7. Dalrymple BP, Peters JM, Böse R, Wright IG. A polymerase chain reaction method for the identification of genes encoding members of the Bv60/p58 family of rhoptry protein homologues in the genus Babesia.. Exp Parasitol 1996 Oct;84(1):96-100.
                    pubmed: 8888737doi: 10.1006/expr.1996.0094google scholar: lookup
                  8. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.. Anal Biochem 1983 Jul 1;132(1):6-13.
                    pubmed: 6312838doi: 10.1016/0003-2697(83)90418-9google scholar: lookup
                  9. Friedhoff KT. [Piroplasmas of horses--impact on the international horse trade].. Berl Munch Tierarztl Wochenschr 1982 Oct 1;95(19):368-74.
                    pubmed: 6756387
                  10. Honda Y, Waithaka M, Taracha EL, Duchateau L, Musoke AJ, McKeever DJ. Delivery of the Theileria parva p67 antigen to cattle using recombinant vaccinia virus: IL-2 enhances protection.. Vaccine 1998 Aug;16(13):1276-82.
                    pubmed: 9682391doi: 10.1016/s0264-410x(98)00041-3google scholar: lookup
                  11. Ikadai H, Kabamoto S, Xuan X, Igarashi I, Nagasawa H, Fujisaki K, Suzuki N, Mikami T. Protein analysis of Babesia caballi merozoites by two-dimensional polyacrylamide gel electrophoresis and western blotting.. J Vet Med Sci 2000 Mar;62(3):323-7.
                    pubmed: 10770608doi: 10.1292/jvms.62.323google scholar: lookup
                  12. Ikadai H, Tamaki Y, Xuan X, Igarashi I, Kawai S, Nagasawa H, Fujisaki K, Toyoda Y, Suzuki N, Mikami T. Inhibitory effect of monoclonal antibodies on the growth of Babesia caballi.. Int J Parasitol 1999 Nov;29(11):1785-91.
                    pubmed: 10616924doi: 10.1016/s0020-7519(99)00137-xgoogle scholar: lookup
                  13. Maniatis T, Fritsch E F, Sambrook J. Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory; 1982.
                  14. McElwain TF, Perryman LE, Davis WC, McGuire TC. Antibodies define multiple proteins with epitopes exposed on the surface of live Babesia bigemina merozoites.. J Immunol 1987 Apr 1;138(7):2298-304.
                    pubmed: 2435796
                  15. McElwain TF, Perryman LE, Musoke AJ, McGuire TC. Molecular characterization and immunogenicity of neutralization-sensitive Babesia bigemina merozoite surface proteins.. Mol Biochem Parasitol 1991 Aug;47(2):213-22.
                    pubmed: 1944418doi: 10.1016/0166-6851(91)90181-5google scholar: lookup
                  16. Mishra VS, McElwain TF, Dame JB, Stephens EB. Isolation, sequence and differential expression of the p58 gene family of Babesia bigemina.. Mol Biochem Parasitol 1992 Jul;53(1-2):149-58.
                    pubmed: 1501634doi: 10.1016/0166-6851(92)90017-egoogle scholar: lookup
                  17. Mishra VS, Stephens EB, Dame JB, Perryman LE, McGuire TC, McElwain TF. Immunogenicity and sequence analysis of recombinant p58: a neutralization-sensitive, antigenically conserved Babesia bigemina merozoite surface protein.. Mol Biochem Parasitol 1991 Aug;47(2):207-12.
                    pubmed: 1944417doi: 10.1016/0166-6851(91)90180-egoogle scholar: lookup
                  18. Palmer GH, McElwain TF, Perryman LE, Davis WC, Reduker DR, Jasmer DP, Shkap V, Pipano E, Goff WL, McGuire TC. Strain variation of Babesia bovis merozoite surface-exposed epitopes.. Infect Immun 1991 Sep;59(9):3340-2.
                    pmc: PMC258180pubmed: 1715329doi: 10.1128/iai.59.9.3340-3342.1991google scholar: lookup
                  19. Purnell R E. Babesiosis in various hosts. In: Ristic M, Krier J P, editors. Babesiosis. New York, N.Y: Academic Press, Inc.; 1981. pp. 25–64.
                  20. Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1989.
                  21. Sam-Yellowe TY. Rhoptry organelles of the apicomplexa: Their role in host cell invasion and intracellular survival.. Parasitol Today 1996 Aug;12(8):308-16.
                    pubmed: 15275182doi: 10.1016/0169-4758(96)10030-2google scholar: lookup
                  22. Schein E. Equine babesiosis. In: Ristic M, editor. Babesiosis of domestic animals and man. Boca Raton, Fla: CRC Press, Inc.; 1988. pp. 197–208.
                  23. Short JM, Fernandez JM, Sorge JA, Huse WD. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties.. Nucleic Acids Res 1988 Aug 11;16(15):7583-600.
                    pmc: PMC338428pubmed: 2970625doi: 10.1093/nar/16.15.7583google scholar: lookup
                  24. Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.. Gene 1988 Jul 15;67(1):31-40.
                    pubmed: 3047011doi: 10.1016/0378-1119(88)90005-4google scholar: lookup
                  25. Suarez CE, Palmer GH, Jasmer DP, Hines SA, Perryman LE, McElwain TF. Characterization of the gene encoding a 60-kilodalton Babesia bovis merozoite protein with conserved and surface exposed epitopes.. Mol Biochem Parasitol 1991 May;46(1):45-52.
                    pubmed: 1712911doi: 10.1016/0166-6851(91)90197-egoogle scholar: lookup
                  26. Tanaka S, Yora T, Nakayama K, Inoue K, Kurosumi K. Proteolytic processing of pro-opiomelanocortin occurs in acidifying secretory granules of AtT-20 cells.. J Histochem Cytochem 1997 Mar;45(3):425-36.
                    pubmed: 9071324doi: 10.1177/002215549704500310google scholar: lookup
                  27. 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
                  28. 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
                  29. Wright IG, Casu R, Commins MA, Dalrymple BP, Gale KR, Goodger BV, Riddles PW, Waltisbuhl DJ, Abetz I, Berrie DA. The development of a recombinant Babesia vaccine.. Vet Parasitol 1992 Sep;44(1-2):3-13.
                    pubmed: 1441189doi: 10.1016/0304-4017(92)90138-ygoogle scholar: lookup
                  30. Yasuda A, Kimura-Kuroda J, Ogimoto M, Miyamoto M, Sata T, Sato T, Takamura C, Kurata T, Kojima A, Yasui K. Induction of protective immunity in animals vaccinated with recombinant vaccinia viruses that express PreM and E glycoproteins of Japanese encephalitis virus.. J Virol 1990 Jun;64(6):2788-95.
                    pmc: PMC249459pubmed: 2159544doi: 10.1128/jvi.64.6.2788-2795.1990google scholar: lookup

                  Citations

                  This article has been cited 17 times.
                  1. El-Sayed SAE, Rizk MA, Baghdadi HB, Ringo AE, Sambuu G, Nugraha AB, Igarashi I. Development of a promising antigenic cocktail for the global detection of Babesia caballi in horse by ELISA.. PLoS One 2023;18(4):e0284535.
                    doi: 10.1371/journal.pone.0284535pubmed: 37058508google scholar: lookup
                  2. Yang G, Zhou B, Chen K, Hu Z, Guo W, Wang X, Du C. Diagnostic Performance of Competitive ELISA and Western Blot Methods for the Detection of Antibodies against Theileria equi and Babesia caballi.. Microorganisms 2022 Dec 21;11(1).
                    doi: 10.3390/microorganisms11010021pubmed: 36677312google scholar: lookup
                  3. Lv K, Zhang Y, Yang Y, Liu Z, Deng L. Development of Nested PCR and Duplex Real-Time Fluorescence Quantitative PCR Assay for the Simultaneous Detection of Theileria equi and Babesia caballi.. Front Vet Sci 2022;9:873190.
                    doi: 10.3389/fvets.2022.873190pubmed: 35664851google scholar: lookup
                  4. Díaz-Sánchez AA, Pires MS, Estrada CY, Cañizares EV, Del Castillo Domínguez SL, Cabezas-Cruz A, Rivero EL, da Fonseca AH, Massard CL, Corona-González B. First molecular evidence of Babesia caballi and Theileria equi infections in horses in Cuba.. Parasitol Res 2018 Oct;117(10):3109-3118.
                    doi: 10.1007/s00436-018-6005-5pubmed: 30033488google scholar: lookup
                  5. Rodriguez M, Alhassan A, Ord RL, Cursino-Santos JR, Singh M, Gray J, Lobo CA. Identification and characterization of the RouenBd1987 Babesia divergens Rhopty-Associated Protein 1.. PLoS One 2014;9(9):e107727.
                    doi: 10.1371/journal.pone.0107727pubmed: 25226276google scholar: lookup
                  6. 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
                  7. Singh H, Mishra AK, Rao JR, Tewari AK. Comparison of indirect fluorescent antibody test (IFAT) and slide enzyme linked immunosorbent assay (SELISA) for diagnosis of Babesia bigemina infection in bovines.. Trop Anim Health Prod 2009 Feb;41(2):153-9.
                    doi: 10.1007/s11250-008-9170-1pubmed: 19115088google scholar: lookup
                  8. Salim BO, Hassan SM, Bakheit MA, Alhassan A, Igarashi I, Karanis P, Abdelrahman MB. Diagnosis of Babesia caballi and Theileria equi infections in horses in Sudan using ELISA and PCR.. Parasitol Res 2008 Oct;103(5):1145-50.
                    doi: 10.1007/s00436-008-1108-zpubmed: 18618143google scholar: lookup
                  9. Heim A, Passos LM, Ribeiro MF, Costa-Júnior LM, Bastos CV, Cabral DD, Hirzmann J, Pfister K. Detection and molecular characterization of Babesia caballi and Theileria equi isolates from endemic areas of Brazil.. Parasitol Res 2007 Dec;102(1):63-8.
                    doi: 10.1007/s00436-007-0726-1pubmed: 17828553google scholar: lookup
                  10. 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
                  11. Hirata H, Yokoyama N, Xuan X, Fujisaki K, Suzuki N, Igarashi I. Cloning of a novel Babesia equi gene encoding a 158-kilodalton protein useful for serological diagnosis.. Clin Diagn Lab Immunol 2005 Feb;12(2):334-8.
                    doi: 10.1128/CDLI.12.2.334-338.2005pubmed: 15699430google scholar: lookup
                  12. Boonchit S, Xuan X, Yokoyama N, Goff WL, Waghela SD, Wagner G, Igarashi I. Improved enzyme-linked immunosorbent assay using C-terminal truncated recombinant antigens of Babesia bovis rhoptry-associated protein-1 for detection of specific antibodies.. J Clin Microbiol 2004 Apr;42(4):1601-4.
                    doi: 10.1128/JCM.42.4.1601-1604.2004pubmed: 15071011google scholar: lookup
                  13. Tamaki Y, Hirata H, Takabatake N, Bork S, Yokoyama N, Xuan X, Fujisaki K, Igarashi I. Molecular cloning of a Babesia caballi gene encoding the 134-kilodalton protein and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.. Clin Diagn Lab Immunol 2004 Jan;11(1):211-5.
                    doi: 10.1128/cdli.11.1.211-215.2004pubmed: 14715570google scholar: lookup
                  14. Hirata H, Xuan X, Yokoyama N, Nishikawa Y, Fujisaki K, Suzuki N, Igarashi I. Identification of a specific antigenic region of the P82 protein of Babesia equi and its potential use in serodiagnosis.. J Clin Microbiol 2003 Feb;41(2):547-51.
                    doi: 10.1128/JCM.41.2.547-551.2003pubmed: 12574244google scholar: lookup
                  15. Boonchit S, Xuan X, Yokoyama N, Goff WL, Wagner G, Igarashi I. Evaluation of an enzyme-linked immunosorbent assay with recombinant rhoptry-associated protein 1 antigen against Babesia bovis for the detection of specific antibodies in cattle.. J Clin Microbiol 2002 Oct;40(10):3771-5.
                    doi: 10.1128/JCM.40.10.3771-3775.2002pubmed: 12354879google scholar: lookup
                  16. Hirata H, Ikadai H, Yokoyama N, Xuan X, Fujisaki K, Suzuki N, Mikami T, Igarashi I. Cloning of a truncated Babesia equi gene encoding an 82-kilodalton protein and its potential use in an enzyme-linked immunosorbent assay.. J Clin Microbiol 2002 Apr;40(4):1470-4.
                    doi: 10.1128/JCM.40.4.1470-1474.2002pubmed: 11923375google scholar: lookup
                  17. Fukumoto S, Xuan X, Nishikawa Y, Inoue N, Igarashi I, Nagasawa H, Fujisaki K, Mikami T. Identification and expression of a 50-kilodalton surface antigen of Babesia gibsoni and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.. J Clin Microbiol 2001 Jul;39(7):2603-9.
                    doi: 10.1128/JCM.39.7.2603-2609.2001pubmed: 11427577google scholar: lookup
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