Cloning and Expression of a 48-KilodaltonBabesia caballi Merozoite Rhoptry Protein and Potential Use of the Recombinant Antigen in an Enzyme-Linked Immunosorbent Assay

A cDNA expression library prepared from Babesia caballimerozoite 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 theB. 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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A member of the HSP90 family from ovineBabesiain China: molecular characterization, phylogenetic analysis and antigenicity

Parasitology ◽

10.1017/s0031182015000797 ◽

2015 ◽

Vol 142 (11) ◽

pp. 1387-1397 ◽

Cited By ~ 5

Author(s):

GUIQUAN GUAN ◽

JUNLONG LIU ◽

AIHONG LIU ◽

YOUQUAN LI ◽

QINGLI NIU ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Amino Acid ◽

Structural Characteristics ◽

Heat Shock Protein 90 ◽

Cellular Transformation ◽

Amino Acid Sequences ◽

Enzyme Linked Immunosorbent Assay ◽

Cdna Expression Library ◽

Expression Library ◽

Reading Frame

SUMMARYHeat shock protein 90 (HSP90) is a key component of the molecular chaperone complex essential for activating many signalling proteins involved in the development and progression of pathogenic cellular transformation. AHsp90gene (BQHsp90) was cloned and characterized fromBabesiasp. BQ1 (Lintan), an ovineBabesiaisolate belonging toBabesia motasi-like group, by screening a cDNA expression library and performing rapid amplification of cDNA ends. The full-length cDNA ofBQHsp90is 2399 bp with an open reading frame of 2154 bp encoding a predicted 83 kDa polypeptide with 717 amino acid residues. It shows significant homology and similar structural characteristics toHsp90of other apicomplex organisms. Phylogenetic analysis, based on the HSP90 amino acid sequences, showed that theBabesiagenus is clearly separated from other apicomplexa genera. Five Chinese ovineBabesiaisolates were divided into 2 phylogenetic clusters, namelyBabesiasp. Xinjiang (previously designated a new species) cluster andB. motasi-like cluster which could be further divided into 2 subclusters (Babesiasp. BQ1 (Lintan)/Babesiasp. Tianzhu andBabesiasp. BQ1 (Ningxian)/Babesiasp. Hebei). Finally, the antigenicity of rBQHSP90 protein from prokaryotic expression was also evaluated using western blot and enzyme-linked immunosorbent assay (ELISA).

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Molecular Cloning of a Babesia caballi Gene Encoding the 134-Kilodalton Protein and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.1.211-215.2004 ◽

2004 ◽

Vol 11 (1) ◽

pp. 211-215 ◽

Cited By ~ 2

Author(s):

Yoh Tamaki ◽

Haruyuki Hirata ◽

Noriyuki Takabatake ◽

Sabine Bork ◽

Naoaki Yokoyama ◽

...

Keyword(s):

Molecular Cloning ◽

Immunosorbent Assay ◽

Horse Serum ◽

Enzyme Linked Immunosorbent Assay ◽

Gene Encoding ◽

Infected Horse ◽

Babesia Caballi ◽

Babesia Equi ◽

Diagnostic Potential ◽

Control Horse

ABSTRACT A Babesia caballi gene encoding the 134-kDa (BC134) protein was immunoscreened with B. caballi-infected horse serum. An enzyme-linked immunosorbent assay (ELISA) using recombinant BC134 protein could effectively differentiate B. caballi-infected horse sera from Babesia equi-infected or noninfected control horse sera. These results suggest that the recombinant BC134 protein is a potential diagnostic antigen in the detection of B. caballi infection.

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Detection of Babesia caballi infection by enzyme-linked immunosorbent assay using recombinant 48-kDa merozoite rhoptry protein

International Journal for Parasitology ◽

10.1016/s0020-7519(00)00008-4 ◽

2000 ◽

Vol 30 (5) ◽

pp. 633-635 ◽

Cited By ~ 31

Author(s):

Hiromi Ikadai ◽

Claudia Rocio Osorio ◽

Xuenan Xuan ◽

Ikuo Igarashi ◽

Takumi Kanemaru ◽

...

Keyword(s):

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Babesia Caballi ◽

Rhoptry Protein

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Serological Diagnosis of Human Cysticercosis by Use of Recombinant Antigens from Taenia solium Cysticerci

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.6.4.479-482.1999 ◽

1999 ◽

Vol 6 (4) ◽

pp. 479-482 ◽

Cited By ~ 20

Author(s):

Kerstin Hubert ◽

Abel Andriantsimahavandy ◽

Alain Michault ◽

Matthias Frosch ◽

Fritz A. Mühlschlegel

Keyword(s):

Immunosorbent Assay ◽

Fusion Proteins ◽

Taenia Solium ◽

Serological Diagnosis ◽

Enzyme Linked Immunosorbent Assay ◽

Cdna Expression Library ◽

Serum Samples ◽

Expression Library ◽

Recombinant Antigens ◽

Cdna Expression

ABSTRACT A Taenia solium metacestode cDNA expression library in the lambda ZAPII vector was screened with pooled sera from patients with neurocysticercosis. Sixty primary clones were identified and shown to belong to two classes. The clones NC-3 and NC-9 did not reveal any significant homologies to sequences deposited in the databases and were further characterized. Both recombinant antigens were expressed as glutathione S-transferase fusion proteins and applied for serological diagnosis of human cysticercosis. An enzyme-linked immunosorbent assay was established and evaluated with 27 serum samples of La Réunion and Madagascar patients with cysticercosis. Diagnosis in these patients was established with radiological and serological procedures. For antigen NC-3 a sensitivity of 96.3% and a specificity of 91.5% for the serodiagnosis were achieved. In contrast, the sensitivity of antigen NC-9 was only 33.3%.

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Detection of Equine Antibodies to Babesia caballi by Recombinant B. caballi Rhoptry-Associated Protein 1 in a Competitive-Inhibition Enzyme-Linked Immunosorbent Assay

Journal of Clinical Microbiology ◽

10.1128/jcm.37.7.2285-2290.1999 ◽

1999 ◽

Vol 37 (7) ◽

pp. 2285-2290 ◽

Cited By ~ 48

Author(s):

Lowell S. Kappmeyer ◽

Lance E. Perryman ◽

Stephen A. Hines ◽

Timothy V. Baszler ◽

Jonathan B. Katz ◽

...

Keyword(s):

Immunosorbent Assay ◽

Competitive Inhibition ◽

Immunofluorescence Assay ◽

Cloning And Expression ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Gene Encoding ◽

Peptide Epitope ◽

Babesia Caballi ◽

Serum Dilution

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was developed for detection of equine antibodies specific forBabesia caballi. The assay used recombinant B. caballi rhoptry-associated protein 1 (RAP-1) and monoclonal antibody (MAb) 79/17.18.5, which is reactive with a peptide epitope of a native 60-kDa B. caballi antigen. The gene encoding the recombinant antigen was sequenced, and database analysis revealed that the gene product is a rhoptry-associated protein. Cloning and expression of a truncated copy of the gene demonstrated that MAb 79/17.18.5 reacts with the C-terminal repeat region of the protein. The cELISA was used to evaluate 302 equine serum samples previously tested for antibodies to B. caballi by a standardized complement fixation test (CFT). The results of cELISA and CFT were 73% concordant. Seventy-two of the 77 serum samples with discordant results were CFT negative and cELISA positive. Further evaluation of the serum samples with discordant results by indirect immunofluorescence assay (IFA) demonstrated that at a serum dilution of 1:200, 48 of the CFT-negative and cELISA-positive serum samples contained antibodies reactive with B. caballi RAP-1. Four of five CFT-positive and cELISA-negative serum samples contained antibodies reactive withB. caballi when they were tested by IFA. These data indicate that following infection with B. caballi, horses consistently produce antibody to the RAP-1 epitope defined by MAb 79/17.18.5, and when used in the cELISA format, recombinant RAP-1 is a useful antigen for the serologic detection of anti-B. caballi antibodies.

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