Prokaryotic expression and immunogenicity of 56‐kDa protein of Orientia tsutsugamushi strain Karp

Abstract

To express the 56‐kDa protein of O. tsutsugamushi strain Karp, this protein gene was cloned into pET30a(+) before transforming into host bacteria, E. coli Rossetta. Specificity of the recombinant protein was assessed by ELISA using rabbit sera against common members of the order Rickettsiae and 10 other pathogenic bacteria. After IPTG induction, SDS‐PAGE analysis of isolated protein demonstrated a band at approximately 46‐kDa. Western blot and mass spectrometry analysis proved that the recombinant protein was expressed successfully. Specificity analysis demonstrated that all sera were negative, except sera against O. tsutsugamushi strains TA763, TH1817 and Kato, B. quintana, A. phagocytophilum, E. chaffeensis and B. bacilliformis. The purified protein was used to immunize BALB/c mice and polyclonal antisera were harvested. By examination of IFA and ELISA, the highest titer of the polyclonal antibodies reaches 1:1600. The recombinant 56‐kDa protein in the study is valuable for developing a simple and rapid diagnostic test and vaccine for O. tsutsugamushi.

Quantitative Serodiagnosis of Scrub Typhus Using Surface-Enhanced Raman Scattering-Based Lateral Flow Assay Platforms

“…There has been recent significant interest in SERS-based assay using functional nanoparticles, owing to the high sensitivity and multiplex detection capability. − When SERS nanoparticles are exposed to a laser light, the incident electromagnetic field is greatly enhanced at the active SERS sites by localized surface plasmon effects. − The SERS-based lateral flow assay (SERS-LFA) platform − was specially developed in the present study for sensitive diagnosis of scrub typhus. To increase the specificity of the target antibody, an O. tsutsugamushi recombinant protein was used as the capture probe instead of the O. tsutsugamushi bacteria. , This recombinant protein was synthesized using a 56-kDa type-specific antigen containing three major strains. Specific outer membrane proteins of the three most prevalent O. tsutsugamushi strains, namely, Karp, Kato, and Gilliam, were mixed with a 1:1:1 molar ratio, respectively, and were immobilized on the test line of the LFA strip.…”