Lic13341 Case Study

Characterization of LIC13341 and its orthologs. The CDS LIC13341 from L. interrogans Copenhageni genome was cloned without its predicted signal peptide in pET28a expression vector and induced its expression in E. coli BL21. Induction of protein expression was evaluated using 0.5 mM IPTG where recombinant protein (43 kDa) overexpression was remarkably observed (Fig 2A). In order to further characterize LIC13341, polyclonal antibodies were generated in BALB/c mice against r-LIC13341. To check its antigenic property, ELISA was performed with the mouse immune serum raised against r-LIC13341. The immune response against r-LIC13341 showed a titre of around 1:40000 (Fig 2B). This suggests that LIC13341 is an antigenic protein and there is a host
As the CDS LIC13341 is predicted to be conserved lipoprotein of pathogenic Leptospira and the r-LIC13341 is immunogenic in mice, it was interesting to evaluate whether antibodies present in leptospirosis humans or domestic animals positive can detect r-LIC13341 antigen. It is known that Loa22 is one of the major proteins expressed by the pathogenic Leptospira and is one of the antigens that are being recommended for the diagnosis of leptospirosis (Chalayon et al., 2011; Rajapakse et al., 2015). Thus, to generate a comparative data for the detection of r-LIC13341 (LP46) antigen with the leptospirosis positive serum, LIC10191 (Loa22) was cloned without its signal peptide in pET28a vector, overexpressed and purified using Ni-NTA chromatography (Fig S1A). The polyclonal anti-Loa22 generated in rabbit using purified r-Loa22 was tested to detect both r-Loa22 and native-Loa22 by immunoblot before using it for further experiments (Fig S1B). ELISA was conducted to detect r-LIC13341 and r-Loa22 with the leptospirosis humans and cattles’ (bovine) positive fifty serum samples along with its control serum. Equal amount (400 ng) of r-LIC13341 and r-Loa22 were coated in a microtitre plate and were probed with human or bovine leptospirosis positive serum or negative samples (1:100). To use each recombinant protein in the diagnosis of leptospirosis patients, the cut-off OD value for antibody response was calculated as follows: the mean and standard deviation (SD) values were calculated among the normal control group then the cut-off value of mean+2 SD were used (Table 4). Any OD value from each ELISA assay that equaled or exceeded the cut-off value was regarded as positive for leptospirosis infection and sensitivity (%) of the assay was calculated. Specificity (%) of the assay was calculated based on the number of control group below the calculated cut-off. The sensitivity of the serological assay, based on cut-off