Cloning And Expression Of Outer Lipoprotein LipL32 From Leptospira Icterohaemorrhagiae And Its Application In Antibody Detection Of Leptospires

Leptospirosis is a worldwide zoonotic infectious disease which is caused by the pathogenic Leptospira spp., different leptospiral serovars present a variety of clinical manifestations. Currently the microscopic agglutination test (MAT) is the gold standard of serological test, which requires the maintenance of live cultures of leptospires and dark-field microscopy, is laborious and unsafe. However, the outer lipoprotein LipL32 among pathogenic Leptospira spp. has been reported to be conserved and expressed at high level , capable of inducing neutralizing antibodies in hosts, suggesting that the recombinant LipL32(rLipL32) ELISA may exhibit similar performance, regardless of locally predominant serovars, for serodiagnosis of leptospiral infection.In this study, the outer lipoprotein LipL32 gene was amplified from pathogenic Leptospira Icterohaemorrhagiae by PCR. The amplicon was subsequently cloned into vector pET-30a (+), transformed into E.coli competent cells BL21(DE3). Expression of the recombinant protein was confirmed by Western blot, the recombinant protein was further purified by electroelution.Finally, ELISAs established with the rLipL32 protein as a coating antigen were evaluated for the diagnosis of different sera and compared with MAT.1. Application of the purified recombinant protein in testing sera from rabbits infected with leptospira by indirect and sandwiched ELISA. The results were compared with MAT. It was shown that antibodies from 15 rabbits infected with pathogenic leptospires can be detected by the recombinant protein, while antibodies from 7 rabbits infected with sapropytic leptospires were undetectable, indicating that the outer lipoprotein LipL32 gene only existed in pathogenic leptospires in serological studies.2. Application of the recombinant protein for diagnosis of the confirmed leptospirosis sera including paired acute-and convalescent-phase specimens. When the acute-phase sera panel were tested, our indirect ELISA(r32-I-ELISA) and sandwiched ELISA(r32-S-ELISA) detected 3 and 2 specimens as positive respectively, while the imported commercial IgG ELISA kit(D.A.I-ELISA) available detected 2 as positive, 4 as suspicious, in contrast to 2 positive results obtained by MAT. For the convalescent phase specimens, 8 (88.94%) sera were identified as positive by r32-I-ELISA and r32-S-ELISA, while the D.A.I-ELISA detected only 1(11.11%) as positive, and 7 (77.78%) sera samples were suspicious. These data revealed that ELISA was more sensitive for convalescent-phase samples than the D.A.I-ELISA, the detected rate of ELISA was similar to the MAT .3. Specificity of the recombinant protein. Among the 45 probable cases which MAT positive, 32 (71.11%) samples were positive by r32-I-ELISA, 36 (80.00%) samples were positive 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 96.61% (57/59)for 59 specimens (43 healthy specimens and 16 non-leptospirosis patients included tsutsugamushi disease, hemorrhagic fever ) .There were 14 healthy specimens were negative by D.A.I-ELISA. Among non-leptospirosis patients, two specimens were positive by r32-I-ELISA and r32-S-ELISA, D.A.I-ELISA identified one positive specimen, while 12 specimens were suspicious by D.A.I-ELISA. For 10 syphilis specimens, the data obtained by three ELISAs were in consistent with that by MAT, proving the high specificity of the recombinant LipL32 antigen, it can be used for the screening in large scales field seroepidemiological studies.4. A sandwiched ELISA with the rLipL32 protein as the antigen was developed to detect rat sera. Considering MAT as the standard test, the sensitivity was 86.75%(131/151), the specificity was 99.19%(122/123)and the coincidence rate was 92.34% (253/274). The results revealed that our sandwiched ELISA has similar sensitivity and specificity to that of MAT. In summary, the novel ELISA with rLipL32 has the following advantages in the diagnosis of leptospirosis: faster and easier to perform, particularly suitable for large scales field seroepidemiological studies.