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[An indirect ELISA for the detection of Babesia caballi in equine animals]
Zhen-Li Gong1, Guang-Yuan Liu, Jun-Ren Xie
1Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
Objective:
To clone and express BC48 gene of Babesia caballi, and to establish an indirect ELISA for the diagnosis of B. caballi in equine animals.
Method:
The genomic DNA of B. caballi was extracted from the infected donkey blood. BC48 gene was amplified by PCR. The PCR product was cloned into expression plasmid pET28a, and expressed in E. coli BL21 with IPTG induction. The recombinant protein was purified by Ni-NTA affinity chro-matography and was used as a diagnostic antigen to establish an indirect ELISA. The reaction conditions of the indirect ELISA were optimized. Specificity and sensitivity of this method were evaluated.
Result:
BC48 gene of B. caballi was 1 272 bp. The recombinant protein was expressed in E. coli BL21 as a soluble protein with a molecular weight of about M, 46 000 under induction of IPTG. The concentration of purified protein was 12.98 mg/ml. The best conditions were obtained for the ELISA when the antigen concentration was 65 microg/ml with the serum dilution of 1:80. The protein specifically reacted with serum from donkey infected by B. caballi, but did not react with serum from donkey infected by Theileria equi (B. equi). Both ELISA and microscopy were applied to examine 17 donkeys in the field, 3 were positive by ELISA and 2 were found parasite-positive, respectively.
Conclusion:
The indirect ELISA method may be used to detect B. caballi infection in equine animals.
