Molecular cloning and expression of Rickettsia tsutsugamushi genes for two major protein antigens in Escherichia coli

Abstract

Several polypeptide antigens of Rickettsia tsutsugamushi are recognized by human or primate convalescent sera and may be important protective immunogens. Molecular cloning and expression of the genes encoding the 110K (110 kilodalton) and 56K polypeptide antigens of R. tsutsugamushi Karp were accomplished in the Agtll expression vector system. Southern blot analysis with the cloned fragments for the 56K polypeptide antigen (0.7 kilobases) and the 110K polypeptide antigen (5.4 kilobases) confirmed that the insert DNA was rickettsial and not host cell in origin. Expression of a complete 110K polypeptide was shown to be independent of isopropyl-,3-D-thiogalactopyranoside induction, suggesting that an intact rickettsial promoter was operational. Epitopes of the 56K polypeptide were expressed as lac promoter-dependent ,-galactosidase fusion proteins. Polyclonal antibody, affinity purified against the recombinant 110K and 56K polypeptides, reacted with polypeptides of similar size in the Kato and Gilliam strains of R. tsutsugamushi. Group-reactive, but not strain-specific, monoclonal antibodies against the 56K polypeptide reacted with the cloned portion of the 56K polypeptide. Western blot analysis demonstrated that the cloned 56K Karp antigen gene product is recognized by human convalescent serum.

Immunohistochemical Study of Scrub Typhus: A Report of Two Cases

“…They express epitopes of the 150 kDa, 110 kDa, 72 kDa, 58 kDa, 56 kDa, 49 kDa, 47 kDa and 20 kDa polypeptide antigens [6]. The principal cell wall constituents of O. tsutsugamushi comprise the major strain variable 56-kDa protein as well as the antigenically variable 110-kDa, 47-kDa and 20-kDa proteins [6][7][8][9][10]. We developed a monoclonal antibody recognizing the 47 kDa antigen, which is a surface protein of O. tsutsugamushi.…”

Induction of Protective Immunity Against Scrub Typhus With a 56-Kilodalton Recombinant Antigen Fused With a 47-Kilodalton Antigen of Orientia Tsutsugamushi Karp

“…28−30 Even though the protection against the heterologous strain is not as longlasting as that against the homologous strain, there is some degree of resistance to heterologous challenge, indicating that the cross-reactive antigens of scrub typhus strains may play some roles in protective immunity against heterologous strains of O. tsutsugamushi. 28,30 Both the 56-kD and 47-kD surface proteins of O. tsutsugamushi are recognized as major protective antigens with group-and strain-specific epitopes, which may induce both homologous and heterologous protective immunity against scrub typhus. Based on this reasoning, a fusion protein of Sta56 and Sta47 should be a rational candidate for the development of a more efficacious subunit vaccine.…”

“…However, the group-and strain-specific epitopes of Sta56 and Sta47 were well recognized in previous studies. 11,20,28 Whether Sta56-47 has an ability provide efficient protection against scrub typhus caused by heterotypic strains remains to be demonstrated.…”

Sensitivity of Polymerase Chain Reaction Assay for Rickettsia tsutsugamushi in Patients' Blood Samples

“…Kelly et al have recently reported a PCR method to detect R. tsutsugamushi genome in infected murine tissue (6) . The target sequence was that of the 56-kDa type-specific antigen (12) . PCR products were detectable equally among Gilliam , Karp, and Kato strains.…”