Abstract:

Objective To clone and express the metacaspase gene of Babesia caballi (BcMC), identify its reactivity, and perform bioimformatic analysis. Methods Primers for amplifying the Bcmc gene sequence were designed and synthesized based on previously determined partial gene sequences of B. caballi. The Bcmc gene was then amplified by PCR, and was cloned into the prokaryotic expression vector pET-28a. After extraction of the plasmid pET-28a-Bcmc, double enzyme digestion, PCR, and sequencing were performed for identification. The recombinant plasmids were transformed into competent cells Escherichia coli BL21 (DE3). After optimizing the induction conditions, the optimal IPTG concentration, induction temperature, and time for induction were selected. The expression of the recombinant protein was analyzed by 12% SDS-PAGE. Following purification of the recombinant protein using a His-tag protein purification kit, the reactivity of the recombinant protein was assessed by Western blotting using positive serum from B. caballi infection as the primary antibody. Bioinformatics online software such as ProtParam was utilized to predict the physicochemical properties, phosphorylation sites, subcellular localization, antigenic epitopes, secondary and tertiary structures, and protein interaction networks of the Bcmc gene. The tertiary structure of BcMC was compared with those of Plasmodium spp. MC-1 (PsMC-1) and Trypanosoma theileri MC (TtMC). The Bcmc sequence is being compared using BLAST alignment on the NCBI database. Using Mega 7.0 software, the neighbor-joining method was employed to construct a phylogenetic tree based on the mc gene sequences. Results The PCR amplification product size of the Bcmc gene was approximately 996 bp, consistent with the expected fragment. Identification through double enzyme digestion, PCR, and sequencing of the recombinant plasmid pET-28a-Bcmc indicated the correct insertion of the target gene. The optimization results of induction conditions showed that the expression level of BcMC recombinant protein was highest when the final concentration of IPTG was 0.8 mmol/L and cultured at 37 ℃ for 5 hours. SDS-PAGE results showed that the recombinant protein was expressed in the form of inclusion bodies, with a relative molecular weight of approximately 36 000. Western blotting results demonstrated that the purified BcMC could specifically react with positive serum from B. caballi infection. Bioinformatics analysis revealed that the relative molecular mass of BcMC was 36 956.72 by amino acid physicochemical properties analysis. Phosphate site prediction showed 25 phosphorylation sites for BcMC. Predicted subcellular localization of BcMC in mitochondria accounted for 10%. B-cell antigenic epitope analysis identified 12 potential antigenic epitopes in the protein; the secondary structure of BcMC protein comprised 50.30% irregular coils and 23.19% α-helices. The tertiary structure of BcMC was similar to PsMC-1 and TtMC. Protein interaction network prediction suggested that proteins interacting with BcMC and biological processes involving BcMC were associated with apoptosis. Phylogenetic tree analysis showed that the recombinant plasmid sequences were 99.90% identical to the sequences of Theileria equi (CP099439) and T. equi strain WA (XM 004830992), indicating a close phylogenetic relationship. Conclusion The prokaryotic expressed protein BcMC exhibited good reactivity, and bioinformatics analysis indicated that BcMC is involved in the apoptosis process of B. caballi.

Key words: Babesia caballi, Metcaspase, Horse, Reactogenicity, Bioinformatics analysis