Expression and immune recognition of the conserved MSP4 outer membrane protein of Anaplasma marginale

Abstract

Defining conserved, protective epitopes is essential to the design of an effective vaccine against bovine anaplasmosis. MSP4, one of six initial body proteins recognized by a neutralizing serum, is conserved among Anaplasma mainale isolates at both the protein and the DNA levels. Sera from cattle immunized with an outer membrane fraction ofA. malginale and protected from a virulent challenge bind MSP4. The gene for MSP4 has been cloned, and the recombinant protein has been expressed, isolated, and demonstrated to share epitopes with the native protein expressed on initial bodies. MSP4 may have a greater potential to protect cattle from a challenge by heterologous isolates than other A. marginake surface proteins, which vary widely in size and structure.

The immunoprotective Anaplasma marginale major surface protein 2 is encoded by a polymorphic multigene family

“…A probe representing the single-copyA. marginale msp-4 gene was used as a control (24). Prehybridization and hybridization were done as previously described (24).…”

Intermolecular relationships of major surface proteins of Anaplasma marginale

“…The Florida strain of A. marginale induces postinfection immunity against heterologous challenge (9,14,28,34) and has been used to identify surface proteins for vaccine development. Four major surface proteins (MSPs) from the Florida strain were recognized by neutralizing polyclonal antibody (23) and designated MSP-1 (105 kDa), MSP-2 (36 kDa), MSP-3 (86 kDa), and MSP-4 (31 kDa) (4,13,17,20,22,24). MSP-5 was originally identified in outer membrane preparations of the Norton Zimbabwe strain and subsequently shown to be conserved in other strains, including Florida (31).…”

Anaplasma marginale major surface protein 3 is encoded by a polymorphic, multigene family

“…Digestions of genomic A. marginale DNA (1.0 g) and pBluescript msp3-12 (0.1 g) were performed according to the manufacturer's specifications. Digests were Southern blotted under prehybridization and hybridization conditions of 65ЊC in 6ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5ϫ Denhardt's solution (1ϫ Denhardt's solution is 0.02% [wt/vol] Ficoll, 0.02% [wt/vol] polyvinylpyrrolidone, and 0.02% [wt/vol] bovine serum albumin), 0.5% (wt/vol) SDS, and 200 g of sheared herring sperm DNA per ml as previously described (17). Membrane washes (three times for 30 min each) were performed under highly stringent conditions at 65ЊC in 0.5ϫ SSC-0.1% SDS.…”