Cloning and expression of Leptospira strain 017 hemolysin HlyX gene and its cytotoxicity

AIM: To clone HlyX gene and observe its expression and cytotoxicity.METHODS: The HlyX gene was amplified from Leptospira strain 017 genome by the polymerase chain reaction (PCR) and through enzyme digestion, and cloned into pET32a(+), then transformed into E.coliJM109. After induced with IPTG, the target protein was immunized to New zealand white rabbit. Western blotting was used to identify the immunogenicity of the expressed protein. The purified and renatured protein was acted on ECV304 cells to detect its cytotoxicity by examining the release of LDH and NO from the cells. RESULTS: The full length of the HlyX gene about 1 179 bp was obtained by PCR. The recombinant plasmid was identified by enzyme digestion, PCR and DNA sequencing. After induced with IPTG, the expressed protein existed in the form of inclusion bodies about 64 kD, which was consistent with the expected size of the fused protein. After immunity, the titre of the multiclonal antibody reached 1∶〖KG-*2〗64 000 by ELISA. Western blotting analysis found a positive band specifically in the target protein position. The release of the LDH and NO in the ECV304 cells treated with HlyX fusion protein showed significant increase compared with the control group (P<0.01). CONCLUSION: HlyX gene is expressed successfully in E.coli JM109, and the expressed products shows cytotoxicity.