HRP-conjugated goat antirabbit IgG (Dingguo Biotechnology, Beijing, China) diluted by 1:10000 was added and incubated for 1 hr at 37°C. The plates were washed four times with PBS before adding diaminobenzidine substrate (Dingguo Biotechnology), 20 M H2SO4 was added to cease the reaction and the OD490nm was measured. A positive control, a negative control and a blank control were always included on each plate. Six BALB/c mice (6–8 weeks of age) were immunized with the purified recombinant protein. For primary immunization, each mouse was s.c.

injected with 50 μg of antigen (recombinant click here 56-kDa protein) emulsified in Freund’s complete adjuvant. Ten days later, they were given an i.p. booster injection of RXDX-106 in vivo 50 μg antigen emulsified in Freund’s incomplete adjuvant. Control mice were injected similarly with PBS emulsified in Freund’s complete adjuvant or incomplete adjuvant. After that, mice were bled and sera were obtained and stored at −20°C. The animal use was reviewed and approved by the Beijing Administrative Committee for Laboratory Animals and the animal care met the standard of the committee. Bleeding of the mice was performed by tail clip after primary immunization and cardiac puncture after booster immunization. To determine

IgG titers of the sera, an IFA test with antigen slides of O. tsutsugamushi Karp was carried out with fluorescein isothiocyanate-conjugated goat antimouse IgG (Kierkegaard & Perry Laboratories, Gaithersburg, MD,

USA). Meanwhile, an ELISA test was also performed as described above. A fragment of 1107 bp that would yield a 46-kDa His-tagged protein with a deletion of 99 amino acid residues at the N terminal and 64 amino acid residues at the C terminal was amplified by PCR and the product was cloned into pET30a. The resulting recombinant plasmid, designated pET30a-Ot56, was detected by both PCR and restriction enzyme digestion (Fig. 1) and was verified by direct DNA sequencing. Analysis by SDS-PAGE showed that a band approximately at 46 kDa, the expected size those for the truncated protein, was observed in E. coli Rossetta cells transformed with pET30a-Ot56 (Fig. 2a). The purified protein appeared as a single band corresponding to the molecular mass of the recombinant protein on SDS-PAGE (Fig. 2b). The amount of protein after purification was 0.7 mg/mL. Immunoblot assay showed that the protein was recognized by O. tsutsugamushi Karp-immunized rabbit serum (Fig. 2c). The recombinant protein was also validated by MALDI-TOF-MS, which revealed that it had 100% identity to 56-kDa protein of O. tsutsugamushi (Fig. 3). Enzyme-linked immunoassay was performed to assess the extent of cross-reactivity of the recombinant protein with the rabbit polyclonal sera described above. All of the sera detected, except sera against O. tsutsugamushi strains TA763, TH1817, Kato, B quintana, A. phagocytophilum, E. chaffeensis and B. bacilliformis were negative (Tables 1,2).