Cloning, Expression Of Truncated 56kD Antigen Gene Of Orientia Tsutsugamushi And Its Application With Purified Antigen In Antibody Detection For Scrub Typhus Infection

Objective: To construct recombinant plasmids containing the truncated gene of the major surface antigen Sta56 of Orientia tsutsugamushi (Or.) Karp strain and express the antigen in E.coli . The purified recombinant antigen would be employed in indirect ELISA and rapid immunochromatographic assay with colloidal gold conjugation to detect antibody for scrub typhus infection.Methods: The ORF of sta56 and a 1053bp fragment of the ORF were amplified from the genomic DNA of Orientia tsutsugamushi Karp strain by PCR, then the 1053bp fragment was cloned into T-vector. From the recombinant plasmid TOPO-sta56, several truncated genes of sta56 with different length were amplified and subcloned into the expression vectors pPROEX HTb and pET30a. These genes were expressed in E.coli DH5 a and BL21(DE3) respectively when induced by IPTG. The expressed recombinant proteins were analyzed by SDS-PAGE and Western blot. The recombinant proteins were purified by electroelution or Ni-NTA affinity chromatography. The purified recombinant antigen was applied in an indirect ELISA by coating the polystyrene 96 well plate to detect IgG or conjugated with colloidal gold as the capture reagent in an immunochromatography assay to detect IgM and IgG in Orientia tsutsugamushi infection. Results:1 . The ORF of sta56 was amplified from the genomic DNA of Orientia tsutsugamushi Karp strain and a large fragment (1053bp) of the ORF was successfully cloned into T-vector.2. Six recombinant plamids containing truncated sta56 genes of different length were constructed as follow: pHTbOt957, pHTbOt498, pHTbOt342and pETOt957, pETOt498, pETOt342. The recombinant Sta56 proteins were highly expressed as 6XHis fusion proteins in E.coli DH5 a and BL21(DE3) respectively. The fusion proteins showed correct bands while analysed with SDS-PAGE and western blot with the positive serum from Ot. patients.3. Ni-NTA affinity chromatography and electroelution were used to purify the recombinant protein and satisfactory purity for diagnostic reagent was achieved.4. An indirect ELISA employing the recombinant protein as coating antigen to detect IgG produced in Ot. infection showed 91.7% in sensitivity and 76.5% in specificity, in comparing with IFAT.5. The immunochromatographic assay applying the purified recombinant antigen conjugated with colloidal gold as the capture reagent can detect IgM and IgG from Orientia tsutsugamushi infection in the same time, with 94.2% in sensitivity and 84.2% in specificity in IgG detection, in comparing with IFAT.Conclusion: The sta56 gene of Orientia tsutsugamushi Karp strain could be highly expressed in E. coli. The recombinant Sta56 antigen with immunoreactivity could be used as diagnostic reagent for Ot. infection. The indirect ELISA is suitable for epidemiological investigation while the immunochromatographic assay can be applied in quick diagnosis for clinical patients in the countryside or hospitals without basic experimental equipments.