Expression, purification and identification of 56kDa protein of orientia tsutsugamushi in prokaryotic cells

  Objective   To express and purify the 56kDa protein of orientia tsutsugamushi (Ot) Gilliam strain in escherichia coli to obtain the abundant recombinant protein with high activity and to play a foundation for preparing the diagnosis kit of scrub typhus.

  Methods   The gene (about 1 320 bp) encoding 56kDa protein of Ot was amplified by using PCR technique. PCR product was cloned into the PET 28a expression vector, plasmid PET-OTG was constructed and expressed successfully. The recombinant protein containing sixtag was purified by Ni²⁺ chromatography column. The positive fractions with significant amounts of protein were analyzed by SDS-PAGE and identified both by western-blot and indirect ELISA.

  Results   The recombinant plasmid containing the aimgene was expressed successfully both by forming inclusion body and soluble protein, the expressed fusion protein was visualized on gel at molecular mass about 52 kDa. The inclusion body protein can get a higher purity than soluble protein after being purified by chromatography column. By western-blot the recombinant proteins can be recognized by patient's positive serum while the negative serum cannot resulted in the same blot band. By indirect ELISA, the recombinant proteins showed good antigenicity that can distinguish the positive and negative serum successfully.

  Conclusion   The purified recombinant protein with immuno-reactivity may be a promising diagnostic antigen for preparing the diagnosis kit of tsutsug amushi disease.