Fig 1.
Schematic representation of the genes optimized for the expression of the truncated Lci2, Lci3 and Lci12 proteins and evaluation with human and canine sera.
(A) Sequence of the repeats chosen to be included in the proteins encoded by the three synthetic genes. (B) Scheme detailing the organization of the optimized genes coding for the truncated variants of the three proteins selected for this study. The various regions with and without repeats are indicated, as well as relevant sites for the restriction enzymes used in cloning/subcloning procedures. (C) ELISA showing the evaluation of the different recombinant proteins described in the text with human and canine sera from VL afflicted individuals (VL) and healthy controls (Ctrl). NT (N–Terminal region) and CT (C–Terminal region).
Fig 2.
Preliminary assessment of a first set of chimeric proteins based on the Lci2, Lci3 and Lci12 antigens.
(A) Schematic representation of the three chimeric proteins generated through subcloning of the Lci2, Lci3 and Lci12 synthetic gene fragments, as described in the text. The chimeric constructs differ in the order in which the different gene fragments are placed along the protein synthesis as well as in their C-terminal end (Lci2 for D1; Lci3 for D2 and D3) (the full-length DNA and protein sequences of the D2 chimera are shown in the Supporting S9 and S10 Figs). (B) SDS-PAGE gel evaluation of the corresponding chimeric proteins after expression in Escherichia coli and affinity purification. Numbers on the left indicate the sizes of molecular weight markers, with the red arrows highlighting the purified recombinant proteins. (C) ELISA comparing the recognition of the three chimeric proteins by human sera from VL afflicted and control individuals. CT (C-Terminal region).
Table 1.
Sensitivity, specificity and accuracy values for the different chimeric proteins evaluated in this study with both sets of human and canine sera.
Fig 3.
Schematic representation of the in silico designed chimeric genes evaluated here.
(A) Representation of the four in silico designed chimeric genes originally synthesized (Q1 through Q4), indicating the fragments derived from the selected antigens as well as relevant restriction sites for further cloning procedures. (B) Representation of the constructs derived from the Q1 gene after removal of selected fragments through digestion with restriction enzymes and religation. Q1NN: Q1 after removal of the pSS-gIII tag through digestion with NcoI and religation; Q1SX: Q1 after removal of the Lci13-CT fragment, through digestion with SalI/XhoI and religation. CT (C-Terminal region), A (Antigenic region).
Fig 4.
Expression of the in silico designed chimeric proteins in E. coli.
(A) Representative Coomassie-Blue stained SDS-PAGE gel evaluating the expression of fourteen of the proteins encoded by the in silico designed chimeric genes. These were assessed in whole extracts of IPTG induced Rosetta 2 Escherichia coli cells. (B) Western blot assay assaying the recognition of selected chimeric proteins in whole E. coli extracts with a commercial anti-histidine monoclonal antibody. (C) Western blot evaluating the recognition of selected chimeric proteins with poly-clonal sera raised against the Lci3, Lci12 and Lci13 recombinant proteins. The red arrows highlight the bands corresponding to the expected sizes for the different chimeric proteins.
Fig 5.
Serological evaluation of selected chimeric proteins.
(A) SDS-PAGE gel showing the affinity purification of the proteins with best performances in expression in Escherichia coli. (B) and (C) Summary of the ELISA results and ROC curve analyses (top and bottom panels, respectively) of the chimeric proteins with sera from dogs and humans with VL, as well as healthy control individuals.
Fig 6.
Design and evaluation of the new chimeric Q5 protein.
(A) Schematic representation of the new Q5 construct designed with the extra Lci3 repeats (in comparison with Q1SX) flanked by Sal I/Xho I restriction sites. (B) SDS-PAGE and Western-blot evaluation of the Q5 recombinant protein. The left panel shows the Coomassie-Blue stained SDS-PAGE gel evaluation of the Q5 expression in whole cell bacterial extracts. The middle panel shows a western blot comparing the recognition of Q1SX and Q5 in whole bacterial extracts by an anti-his antibody, with the right panel confirming the affinity purification of the Q5 protein in another Coomassie-Blue stained gel. (C) ELISA assay evaluating the Q5 protein with both human and canine sera from VL afflicted individuals. (D) ROC curve analyses of the Q5 protein proteins with sera from dogs and humans with VL. (E) Same as (C) but comparing the recombinant Q5 with human sera from individuals with tegumentary leishmaniasis (TL) as well as corresponding negative controls.