ABSTRACT
Objective To establish recombinant outer membrane lipoprotein LipL32-based antibody detection assays in identifying leptospirosis. Methods Recombinant leptospiral outer membrane protein LipL32 was obtained by genetic engineering method. This purified protein was used in the indirect and sandwich ELISA assays to test the antibodies in sera of human beings and rats, and the results were compared with those obtained by microscopy agglutination test (MAT) and imported ELISA kit. Results When the acute and convalescent phase specimens from 9 leptospiral patients were tested, the detected rates of three ELISAs were similar to the MAT. Among the 45 probable cases which MAT showed positive, 32 (71.11%) samples were positive by r32-I-ELISA, 36(80.00%) by r32-S-ELISA,while 28.89% (13/45) samples were positive and 55.56% (25/45)were suspicious by D.A.I-ELISA. The specificity of r32-I-ELISA and r32-S-ELISA were 97.10 % (67/69) for 69 specimens. 43 healthy specimens were negative by both r32-I-ELISA and r32-S-ELISA, 14 healthy specimens were negative by D.A.I-ELISA. Among 16 non-leptospirosis patients, two specimens were positive by r32-I-ELISA and r32-S-ELISA, D.A.I-ELISA and identified one positive specimen, while 12 specimens were suspicious by D.A.I-ELISA. For 10 syphilis specimens, data obtained through three ELISAs were in consistent with that by MAT. A sandwiched ELISA, using rLipL32 protein as the antigen was developed to detect rat sera. Considering MAT as standard test, the sensitivity and specificity were 86.75 % (131/151), 99.19 % (122/123) respectively with coincidence rate as 92.34% (253/274). Conclusion The recombinant protein LipL32 had high immunoresctivity and could be used as an antigen for the detection of panthogenic leptospirosis. In summary, the novel sandwiched ELISA with rLipL32 showed similar sensitivity and specificity to that of MAT in the antibody detection of rat leptospirosis. It was suitable for large scales field sero-epidemiological studies.
ABSTRACT
<p><b>OBJECTIVE</b>To construct recombinant plasmids containing the truncated gene of the major surface antigen sta56 of Orientia tsutsugamushi (Ot.) Karp strain for expression antigen in E. coli so as to compare the expression efficiency in different systems.</p><p><b>METHODS</b>From the recombinant plasmid TOPO-sta56 containing sta56 of Orientia tsutsugamushi Karp strain, 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 DH5alpha and BL21(DE3) respectively when induced by IPTG. The expressed recombinant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.</p><p><b>RESULTS</b>Six recombinant plasmids containing truncated sta56 genes of different length were constructed as follow: pHTbOt957, pHTbOt498, pHTbOt342 and pETOt957, pETOt498, pETOt342. The recombinant sta56 proteins were highly expressed as 6 x His fusion proteins in E. coli DH5alpha and BL21(DE3) respectively. The fusion proteins showed as different bands of different molecular weight respectively when analyzed with SDS-PAGE. Western blot demonstrated that the recombinant proteins were recognized by the positive serum of Ot. patients.</p><p><b>CONCLUSION</b>The sta56 gene of Orientia tsutsugamushi Karp strain could be highly expressed in E. coli and its expression showed better efficiency in pET30a than in pPROEX HTb. The recombinant sta56 antigen with immunoreactivity could be used as diagnostic reagent for Ot. infection.</p>