[Recombinant plasmid constructed and cytotoxicity studied for outer membrane protein LipL32 gene of Leptospira strain 017].

ABSTRACT

OBJECTIVE: To construct the recombinant plasmid containing the outer membrane protein LipL32 gene of Leptospira strain 017 and to study on the cytotoxicity of the expression protein. METHODS: By the polymerase chain reaction (PCR), the LipL32 gene was amplified from Leptospira strain 017 genome and cloned into pET32a(+) with enzyme digestion, then used to transform E. coli JM109. After induced with IPTG, the target protein was expressed and used to immunize New zealand white rabbit. Western Blotting identified the immunogenicity of the expressed protein. Then the purified and renatured protein was acted on ECV304 cell so as to get its cytotoxicity detected by examining the LDH and NO (nitrogen monoxide) release from cell. RESULTS: The full length of the LipL32 gene about 816 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 mainly in the form of inclusion bodies about 52 x 10(3) (relative molecular mass) which was consistent with the expected size of the fused protein. After rabbit immunity, the titre of the produced multiclonal antibody reached 1 32 000 measured by ELISA. Western Blotting analysis found a positive band specifically in the target protein position. The release of the LDH and NO of the ECV304 cell treated with LipL32 had significant increase compared with the control group. CONCLUSION: The recombinant plasmid containing LipL32 gene is successfully constructed and can express the target protein in E. coli JM109. The expressed target protein has cytotoxicity.