High-level expression of a 56-kilodalton protein gene (bor56) of Rickettsia tsutsugamushi Boryong and its application to enzyme-linked immunosorbent assays

Abstract

The 56-kDa protein of Rickettsia tsutsugamushi, which is located on the rickettsial surface, has been shown to be an immunodominant antigen. The gene that encodes the 56-kDa protein of R. tsutsugamushi Boryong (bor56) was cloned. Sequencing revealed an open reading frame of 1,602 bp encoding 534 amino acids with a molecular weight of 56,803. The 56-kDa protein ofR. tsutsugamushi Boryong (Bor56) was expressed as a fusion protein with the maltose-binding protein of Escherichia coli by deleting 252 bp from the 5' end of the open reading frame and subcloning it into the StuI site of pIH821. The recombinant fusion protein was purified by amylose column chromatography for application in an enzyme-linked immunosorbent assay to evaluate the ability of the method to detect the antibody to R. tsutsugamushi in human patient sera. By using sera from 100 patients with scrub typhus and 70 patients with other febrile diseases, a high diagnostic sensitivity (95%) and a high diagnostic specificity (100%) were demonstrated, suggesting the suitability of the recombinant antigen for use as an immunodiagnostic tool.

Demonstration of Antigenic and Genotypic Variation in Orientia tsutsugamushi Which Were Isolated in Japan, and Their Classification into Type and Subtype

“…However, the strains could be divided into two subtypes, KR-1 and KR-2, by RFLP. The Boryong strain was isolated in Korea (2) and the 56-I(Da protein gene was cloned and sequenced by Kim et al (11). They reported that the 56-kDa protein of this strain showed 99.1% homology at the amino acid sequence level (5 amino acid replacements in 532 residues) with that of Kuroki strain (prototype), and the theoretical RFLP patterns of the Boryong strain generated by computer analysis with a program for restriction mapping were identical with those of Kuroki strain in all 5 endonuclease digestions, indicating that Boryong strain should be included in the KR-1 subtype.…”

Phylogenetic analysis of Orientia tsutsugamushi strains based on the sequence homologies of 56-kDa type-specific antigen genes

“…The sequences of 56-kDa genes of six strains, Gilliam, Karp, Kato, Kawasaki, Kuroki and Shimokoshi, were reported previously [9^11]. Sequences of the genes of Yonchon and Boryong strains isolated in South Korea have been reported in Korean studies [12,13], and the sequences of TA678, 686, 716 and 763 strains obtained in Thailand [4] and the Sxh 951 strain in China were cited from the GenBank. The sequencing of other strains in Table 1 was carried out in this study.…”

Prevalence of Genotypes of Orientia tsutsugamushi in Patients with Scrub Typhus in Miyazaki Prefecture

“…Figure 2 shows the DNA sequences of the 0.6-kb PCR products from the 4 strains in Miyazaki, and from the Karp strain reported by Stover et al (10). The DNA sequences of the PCR products from the Tazume and Hirano strains showed a high degree of homology (98.8%), and these two strains were also highly homologous (99.0%) to the reported sequence of the Boryong strain (6). The DNA sequence of the Yoshimura strain revealed a high degree of homology (96.2%) to the Karp strain (10).…”

“…The antigenic variation of O. tsutsugamushi is dependent on a strain-specific 56-kDa protein (12 encoding this 56-kDa protein antigen have been cloned and sequenced from the Karp (10), Gilliam, Kato, Kawasaki, Kuroki, Shimokoshi (8,9) and Boryong strains (6). To clarify the association of each strain in Miyazaki Prefecture with the representative strains, we sequenced the PCR products from Irie, Hirano, Tazume and Yoshimura strains.…”