Screening of a Babesia bigemina cDNA Library with Monoclonal Antibodies Directed to Surface Antigens

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Abstract
A Babesia bigemina cDNA library prepared in lambda ZAP bacteriophage vector was immunoscreened to detect clones expressing surface-exposed epitopes of B. bigemina. A nonradioactive indirect plaque-lift immunoassay was used to detect the positive clones. The primary antibody consisted of a pooled sample of six monoclonal antibodies (mAb) specific for B. bigemina that recognizes various parasite surface antigens of different molecular mass. Screening of approximately 300,000 plaque-forming units from the lambda ZAP cDNA expression library resulted in the identification of five positive clones. The five recombinant clones were immunoscreened individually with each of the six mAb. All five independently obtained clones consisted of lambda ZAP recombinants expressing B. bigemina components recognized by mAb C2F3G3 and B1B3C4. Restriction enzyme digests of rescued recombinant phagemids showed that only four clones contained B. bigemina cDNA. One clone (lambda ZAP Bbi1) contained an insert of approximately 0.6 kBp whereas the other three clones (lambda ZAP Bbi2, lambda ZAP Bbi3, and lambda ZAP Bbi5) carried a cDNA insert of approximately 1.7 kBp. Immunoblotting of protein extracts from recombinants lambda ZAP Bbi2, lambda ZAP Bbi3, and lambda ZAP Bbi5 with mAb C2F3G3 and B1B3C4 demonstrated the expression of a recombinant B. bigemina polypeptide of 55 kDa in E. coli.

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