Expression and Immunogenicity of Recombinant Immunoreactive Surface Protein 2 of Anaplasma phagocytophilum

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Human granulocytic anaplasmosis (HGA), caused by<jats:named-content content-type="genus-species">Anaplasma phagocytophilum</jats:named-content>, is an emerging tick-borne zoonotic disease throughout the world. The first HGA cases in China were documented in 2008, and the greatest challenge posed by the disease is rapid and accurate diagnosis during the acute phage of illness. In this study, we successfully cloned and expressed an<jats:named-content content-type="genus-species">A. phagocytophilum</jats:named-content>immunoreactive surface protein (major surface protein 2 [MSP2]) and demonstrated that this recombinant protein has natural immunogenicity by Western blotting and enzyme-linked immunosorbent assay (ELISA) using human HGA-positive sera and reference rabbit HGA-positive sera. The rabbit antisera against the recombinant protein also reacted actively with the natural antigen of<jats:named-content content-type="genus-species">A. phagocytophilum</jats:named-content>by immunofluorescence assay (IFA). No cross-reaction was observed between the recombinant protein and rabbit antisera against 10 common members of the order<jats:named-content content-type="genus-species">Rickettsiales</jats:named-content>by ELISA when the sera were diluted more than 1:100. We concluded that the recombinant MSP2 protein exhibited excellent antigenicity and specificity, results that should lay the foundation for the development of a simple and rapid diagnostic reagent and a vaccination for anaplasmosis.</jats:p>

Molecular Analysis of Anaplasma phagocytophilum Isolated from Patients with Febrile Diseases of Unknown Etiology in China

“…The amplification of the 16S rRNA gene was performed using a pair of primers [15] (16S-FD1: 5′-AGA GTT TGA TCC TGG CTC AG-3′ and 16S-RP2: 5′-ACG GCT ACC TTG TTA CGA CTT-3′) at an annealing temperature of 56°C. In addition, a pair of primers targeting the ank A gene (PF, position from 679 nt to 697 nt: 5′-CGTATGGATCACCAGAAAG-3′; PR, position from 2464 nt to 2482 nt: 5′-CATTTGCTTCTTGAGGAGT-3′) were used to identify the isolates because they were specific and unique to A. phagocytophilum [16]. All PCR products were confirmed by commercial sequencing (Shanghai Shengong Biotechnology Co., Shanghai, China) using an ABI 3730 sequencing apparatus (Life Technologies, USA) and a Bigdye Terminator V3.1 sequencing kit.…”