Title : Cloning, expression, and characterisation of Wuchereria bancrofti L1 -recombinant filarial protein
Abstract:
Lymphatic filariasis (LF) is a neglected tropical vector-borne disease that infects an estimated 657 million people in 39 countries. It results in irreversible morbidity and disability in infected cases. Though efforts toward global elimination via mass drug administration are in progress, there is a pressing need for efficient diagnostic tests for surveillance purposes in endemic areas. A diagnostic method needs to be developed to diagnose early-stage LF, which demonstrates the limitations of the current methodology. Using the NCBI database, the Wuchereria bancrofti L1 (WbL1) gene sequence was identified and cloned into a PET-30a(+) plasmid vector, following transformation into competent Escherichia coli BL21 (DE3) cells for IPTG-induction. The produced protein was isolated through sonication and purified by immobilised Ni-NTA metal affinity chromatography (IMAC). The purified recombinant filarial protein was characterised and examined through SDS-PAGE, followed by silver staining, western blotting, and LC-MS to confirm its identity, sequence integrity, and intact molecular mass. The purified WbL1 recombinant filarial protein (WbL1 rFP) has a molecular weight of 17.1 kDa verified by western blot verified the rFP using histidine tag antibody. An intact mass analysis and peptide mass fingerprinting shows 18037 Da molecular mass and 97% sequence coverage.
Keywords: Wuchereria bancrofti, Diagnostic antigen, IPTG induction
