| Scrub typhus is a zoonosis caused by Orientia tsutusgamushi(Ot) and transmitted by trombiculid mites. The typical symptoms of scrub typhus include fever, headache, rash, eschar, and lymphadenopathy. Although the disease can be effectively treated with the antibiotic doxycycline, reinfection and relapses occur frequently. In addition, there is a possibility that antibiotic-resistant strains emerge. As a result of such potential problems, the development of scrub typhus vaccines should be pursued. To date, considerable efforts have been invested in developing a vaccine for scrub typhus, but no vaccines for humans are available at present. It is very urgent to develop novel vaccine against scrub typhus.Both 47- and 56-kDa proteins are considered as the major surface proteins of Ot, which has a potential importance in development of subunit vaccine against scrub typhus. The antigenic uniqueness of the 58-kDa heat shock protein of Ot indicates that this protein may be a potentially protective antigen.The genes encoding 47-kDa, 56-kDa, and 58-kDa proteins were amplified from the genomes of Ot. Karp strain by PCR, and the gene fragments were cloned and sequenced. The gene fragments encoding 47-kDa, 56-kDa, and 58-kDa proteins were linked to the prokaryotic expression vector pQE30, constructing recombinant plasmids pQE30/47, pQESO/56, and pQE30/58. In addition, the recombinant plasmids pQE30/56-47 harboring 47-and 56-kDa protein gene and pQE30/58-47 harboring 47- and 58-kDa protein gene were constructed. The transformants harboring the recombinant plasmids were induced to expresse the recombinant proteins with IPTG The expression products 47-kDa, 56-kDa, and 58-kDa proteins as well as 56-47kDa and 58-47kDa fusion protein were detected by SDS-PAGE and the proteins were verified by immunoblot analysis with antiserum to Ot Karp.To study the immunogenicity of the recombinant proteins, the recombinant proteins purified by affinity chromatograph were used to immunize Balb/c mice. After a booster injection, the IgGs to the recombinant proteins of Ot. Karp were detected in the sera from the immunized mice by ELISA. The antigen-specific spleen cell proliferative responses weredemonstrated in the immunized mice by MTT method. The results suggest that the recombinant proteins are the protective antigens capable to stimulate mice to produce specific humoral and cellular immunoresponses. The animals immunized with 56-47kDa fusion protein exhibited profound humoral and cellular immunoresponses, including higher levels of specific IgG antibodies, higher levels of gamma interferon (IFN-y) and interleukin-2 (IL-2), and stronger proliferation of splenic cells in response to homogenous antigen as compared with mice immunized with 47-kDa, 56-kDa, 58-kDa recombinant protein, or 58-47kDa fusion protein.The mice were challenged with 10 LD5o Ot Karp 14 days after second boost immunization. The mortality of mice immunized with 56-47kDa fusion protein was lower than that of the mice immunized with another recombinant protein after challenge of Ot Karp. The level of protection afforded by 56-47kDa fusion protein was 50%, higher than other groups and significantly higher than the negative controls (P <0.05). The mild pathological changes in lungs, livers, and kidneys were observed in the survival mice immunized with 56-47kDa fusion protein. However, the severe pathological changes in these organs were recognized in the unimmunized mice. These results suggest that the mice immunized with 56-47kDa fusion protein exhibited a more increased resistance to Ot Karp as compared with another recombinant protein-immunized mice. These results strongly suggest that 56-47kDa fusion protein contains antigenic epitopes of the 56- and 47-kDa outer membrane protein of Ot Karp and it is a more suitable candidate for replacing whole-cell antigen of Ot Karp to induce protective immunity against scrub typhus.The genes encoding 47- and 56-kDa outer membrane protein were used to construct the recombinant eukaryotic expression plasmids pcDNA/47, pcDNA/56, and pcDN...
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