High expression of LipL21 protein of Iranian Leptospira interrogans in E. coli, applicable for diagnostic ELISA

Abstract

Leptospirosis is an emerging infectious zoonotic disease caused by pathogenic Leptospira. The disease is more prevalent among farmers in hot and humid areas of Iran. Lack of clear clinical signs have impeded the diagnosis of leptospirosis. In this study, we attempted to produce a recombinant LipL21 protein of Leptospira based on a dominant pattern of Iranian isolates and to evaluate it in ELISA test. One hundred and sixty-two complete sequences of LipL21 available in GenBank until January 1, 2019 were compared. One dominant LipL21 protein pattern was selected. The codon optimised sequence was cloned into the pET32a+ expression vector. Trx-LipL21 fusion protein was induced, purified and confirmed by 10% SDS-PAGE followed Coomassie blue staining and immune blotting. For evaluation of effectiveness of rLip21 in ELISA test, 200 µg rLip21 with Montanide ISA70 adjuvant was injected subcutaneously in rabbits three times. Results showed that rLipL21 protein was highly expressed in 2YT media in presence of 0.1 mM IPTG after 16 hours incubation at 37 oC. Recombinant protein was purified 36 mg per liter using affinity batch formation method by Ni-NTA resin. ELISA with micro plate coated with 250 ng rLipL21 protein demonstrated prominently diffe­rences between test and control groups (P<0.01). The rLipL21 protein produced large amounts of antibodies in the rabbit. The protein was also able to detect high levels of antibody in animals immu­nised with Leptospira vaccine. The rLipL21 might be a good candidate for diagnosis and evaluation of antibody levels against Leptospira.