N. Tebele et al., Cloning, characterization, and expression of a 200-kilodalton diagnostic antigen of Babesia bigemina, J CLIN MICR, 38(6), 2000, pp. 2240-2247
Current serological tests for Babesia bigemina use semipurified merozoite a
ntigens derived from infected erythrocytes. One of the major drawbacks of t
hese tests is that antigen quality can vary from batch to batch. Since the
quality of the antigen contributes to the sensitivity and specificity of se
rological tests, the use of standardized recombinant antigens should ensure
consistency in assay quality. Previously, a 200-kDa merozoite antigen (p20
0) was identified as a candidate diagnostic antigen for use in a serologica
l assay for the detection of B. bigemina antibodies in infected cattle. In
this study, we have cloned, characterized, and expressed p200. A 3.5-kbp cD
NA clone encoding p200 was isolated and shown to be almost full length, lac
king approximately 300 bp at the 5' end. The predicted amino acid sequence
shows that p200 consists of a long, highly charged central repeat region of
an uninterrupted alpha helix, indicative of a fibrous protein. Immunoelect
ron microscopy localized p200 to the merozoite cytoplasm, suggesting that t
he antigen may be a structural protein involved in forming filament structu
res within the cytoskeleton. The 3.5-kbp cDNA was expressed in bacteria as
a fusion protein with glutathione S-transferase (GST), but the yield was po
or. To improve the yield, cDNA fragments encoding antigenic domains of p200
were expressed as fusions with GST, One of these fusion proteins, CIA-GST,
is composed of a 7-kDa fragment of the p200 repeat region and contains epi
topes that react strongly with sera from cattle experimentally infected wit
h B. bigemina. Recombinant C1A-GST should permit the development of an impr
oved enzyme-linked immunosorbent assay for the detection of antibodies agai
nst B. bigemina.