Ching and colleagues have developed a rapid immunochromatographic flow test to detect the anti-O. tsutsugamushi IgG and IgM in patients’ sera for diagnosis of scrub typhus, by employing a Karp r56 protein that contained deletions of 79 and 77 amino acid residues at the N and C terminals, respectively, as the diagnostic antigen (19, 20). Antibodies prepared from serum of patients with scrub typhus tend to recognize this protein in general. Mice immunized with the 56-kDa protein generated neutralizing antibodies and showed increased resistance to homologous O. tsutsugamushi infection (21). These data suggest that it is a favorable diagnostic antigen and
vaccine candidate. In this report, we describe the this website molecular cloning, expression and purification of the 56-kDa protein from O. tsutsugamushi strain Karp and investigate the immunogenicity of the recombinant protein. Primers were designed based on the check details published 56-kDa gene nucleotide sequence (GenBank accession no. M33004.1). The upstream and downstream primers were designed to contain NcoI and XhoI restriction sites, respectively: Ot56-F
(positions 298–316), 5′-AGACCATGGCTCAGGTTGAAGAAGGTA-3′; and Ot56-R (positions 1386–1404), 5′-GTCTCGAGCTAAGTATAAGCTAACCCT-3′. Genomic DNA isolated from O. tsutsugamushi strain Karp was used as a template. PCR was performed in a final volume of 50 μL containing approximately 50 ng DNA, 200 μM each deoxyribonucleotide triphosphate, 10 pmol each primer, 5 μL of 10 × PCR buffer (Mg2+ Plus; TaKaRa Biotechnology, Dalian, China) see more and 0.5 U of Ex-Taq DNA polymerase (Takara Biotechnology). Thermal cycling conditions were as follows: 2 min at 95°C, 2 min at 95°C, followed by 30 cycles of 30 s at 94°C, 30 s at 57°C and 1 min at 72°C. A final step of 10 min at 72°C was added to the last cycle. PCR products were analyzed by 1% agarose gel electrophoresis. pET30a(+) and purified PCR products were digested with restriction enzymes NcoI and XhoI (TaKaRa Biotechnology), then ligated overnight at
16°C. The ligation mixture was initially introduced into E. coli DH5α. The recombinant plasmids were identified by PCR, enzyme digestion and were confirmed by sequencing. The plasmid construct was then transformed into E. coli Rossetta (Novagen, Madison, WI, USA) for expression. Escherichia coli Rossetta containing the appropriate plasmid was cultured at 37°C in LB broth containing kanamycin and chloramphenicol. Cultures were induced at an OD600 of 0.6–0.7 with IPTG to a final concentration of 1 mM, and grown for a further 5 hrs. Cells were then pelleted and resuspended in 50 mM phosphate buffer (pH 7.4). After cell lysis by sonication, cellular debris were eliminated by centrifugation at 8000 g for 15 min at 4°C. The water-soluble fraction of the lysate was collected for purification, as described below. To purify the recombinant protein, the cell lysate, containing protein with six His tags, was filtered through a 0.