Cloning and expression of an outer membrane protein of leptospira, LipL32

Abstract

Leptospira, the etiologic agent of leptospirosis, is divided into two groups, leptospira interrogans, comprising all pathogenic strains, and leptospira biflexa, containing the saprophytic strains. Currently, laboratory diagnosis still depends on detection of antibody. Microscopic agglutination test (MAT) which is a reference method, requires maintaining of live organisms and reasing of results requires well trained laboratory personnel. In addittion to MAT, other serological methods have been used for antibody detection, howerver, false positive with other infectious diseases have been reported. David Haake et al., (2000) have characterized leptospiral outer membrane proteins and demonstrated that LipL32, an outer membrame protein of leptospira was found only in pathogenic leptospires and expressed both in vivo and in vitro. For those reasons, LipL32 was proposed to be a candidate antigen for detection of antibody to leptospira and to be a vaccine that may provide cross protection among pathogenic serovars. In this study, leptospira interrogans serovar bratislava was chosen to be a target for cloning of LipL32 gene since it was the most serovar reported to be responsible for leptospirosis cases in Thailand during 1999-2002. LipL32 gene was amplified by PCR and inserted into a plasmid, pRSETc generating recombinant construct, pRSETC-LipL32. Proper insertion was demonstrated by PCR amplification and DNA sequencing. Protein expression from this construct was fusion protein of hisdine and LipL32(His-LipL32) which was further purified using a nickle affinity column. Purified His-LipL32 protein was preliminary tested for its immunogenicity. Antiserum specific for LipL32 reacted with purified LipL32 using immunoblotting. In addition, patient serum positive for antibody to leptospira also reacted against LipL32 protein whereas none of patient serum positive for antibody to T.pallidum and serum from health volunteer demonstrated positive reaction. However, the positive results were obtained in IgG but not IgM antibody detection. Dipstick assay using LipL32 protein as an antigen for IgG specific antibody detection was also performed. Preliminary data suggested that this protein may be a good candidate since sensitivity and specificity compared with MAT, a reference method, were 100 and 98.33%, respectively. In conclusion, cloning and expression of LipL32 gene were done preliminary data demonstrating the possibility o LipL32 to be an antigen for laboratory diagnosis was obtained.