Molecular and recombinant characterization of major surface protein 5 from Anaplasma marginale

Abstract

Anaplasmosis is a tick-borne disease caused by the intracellular rickettsia Anaplasma marginale, which affects cattle and other ruminants in both tropical and subtropical regions of the world, and also causing tremendous economic losses due to decreasing livestock production. The major surface protein 5 (MSP5) of A. marginale is an immunodominant and highly conserved protein encoding by a single gene. In the present study, the complete full-length of the msp5 coding sequence of A. marginale Thailand strain was cloned and determined at a size of 633 bp. Phylogenetic analysis based on neigh-joining (NJ) method showed that the msp5 sequence Thailand strains were clearly distributed in 3rd clade and conserved when compared with other strains. The results showed 9 haplotypes of the msp5 genes, and the entropy analysis of MSP5 amino acid sequences displayed 92 high entropy peaks with value ranging from 0.198 to 0.845 Additionally, a recombinant MSP5 of A. marginale (rAmMSP5) was over-expressed in the E. coli BL21 Star™ (DE3) host cell, affinity purified, and found in SDS-PAGE at a molecular weight of 26 kDa. The antigenicity of rAmMSP5 (26 kDa) and AmMSP5 (19 kDa) was recognized by rabbit anti-rAmMSP5 antisera and A. marginale-infected cattle sera. Both rAmMSP5 and AmMSP5 were perceived by these sera manifesting that recombinant and native AmMSP5 have conserved epitopes. Immunofluorescence technique using rabbit anti-rAmMSP5 antisera exhibited that the AmMSP5 is distributed on both the membrane and the outside of infected erythrocytes. Therefore, the recombinant MSP5 could be used for the development of immunodiagnostic assays and vaccine purposes for controlling anaplasmosis.