Cloning, expression and purification of toxocara canis recombinant antigens (rTES-26, rTES-32, rTES-120) and development of serodiagnostic test for toxocariasis

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dc.contributor.authorMohamad, Suharni
dc.date.accessioned2014-11-03T02:11:00Z
dc.date.available2014-11-03T02:11:00Z
dc.date.issued2009
dc.descriptionPhDen_US
dc.description.abstractRoutine serodiagnosis of human toxocariasis is based on indirect IgG-ELISA kits which employ native Toxocara canis excretory-secretory (TES) antigen. However, these assays lacked specificity especially when used in tropical countries where multiparasitism are prevalent. In an effort to improve the diagnostic test for this infection, we have developed an IgG4-ELISA assay which uses three recombinant antigens. In this study, recombinant T. canis DNA which encode for rTES-26, rTES-32 and rTES-120 were produced by cloning of open-reading frames (ORF) of the respective genes via reverse-transcriptase-PCR (RT-PCR) using mRNA extracted from a culture of T. canis second stage larvae into PCR2.1 TOPO vector. Sequence analysis revealed that TOPO/TES- 32 and TOPO/TES-120 were 100% similar to the reported sequences in the GenBank, however, TOPO/TES-26 gene fragment had four mutations. After all mutations in TOPO/TES-26 gene fragments had been corrected, TES-26 and TES-32 were subsequently subcloned into a GST-tagged, while TES-120 was subcloned into a His-tagged prokaryotic expression vector; and all constructs were expressed in E. coli BL21(DE3) expression host. The recombinant proteins were subsequently purified under native condition by affinity chromatography using GST and His-Trap resins since these recombinant proteins were abundantly expressed in soluble form. The site-specific protease, Factor Xa, was used to remove GST tag in the TES-26 and TES-32 GST-tagged fusion proteins. Western blot analysis revealed that these recombinant antigens were immunologically reactive and specific. Sera from patients infected with toxocariasis had IgG4 antibodies that recognized these recombinant antigens, while sera from individuals with other infections and from healthy normals did not. When the three recombinant antigens were tested in ELISAs specific for immunoglobulin IgM and IgE classes, and IgG subclasses (IgG1-IgG4), the results clearly showed that only IgG4 assay displayed good specificity. The diagnostic utility of each purified recombinant antigen and rTES-30USM (previously produced in our laboratory) was further evaluated by IgG4-ELISA assay using 242 serum samples which included 30 sera from patients with clinical, haematological and serological evidence of toxocariasis. Both rTES-26 and rTES-32 IgG4-ELISAs demonstrated sensitivity of 80.0%; while rTES-120 IgG4-ELISA showed sensitivity of 93.3.0%, which is similar to that previously reported for rTES-30USM IgG4-ELISA. The sensitivity of rTES-120/rTES-30USM IgG4-ELISA was found to be significantly higher than rTES-26/rTES-32 IgG4-ELISA (p<0.001). However, the mean O.Ds of the 30 toxocariasis samples among the IgG4 assays using the four recombinant antigens were shown not to be significantly different (p>0.05). At p<0.05, there was marginally no significant difference between the specificities of rTES-26 and rTES-120 (p=0.059), rTES-26 and rTES-30USM or between rTES-30USM and rTES-120. In the final assay, rTES-32 was excluded since it was not better than rTES-26 in terms of sensitivity or specificity. Instead rTES-30USM was included due to its high sensitivity and the fact that a 100% detection of toxocariasis cases was achieved when results of IgG4 assays using rTES-30USM and rTES-120 were combined. In summary, a final assay which is sensitive (80% to 93.3%) and specific (92.0%- 96.2%) for detection of toxocariasis was successfully developed using three adjacent wells, each separately coated with rTES-26, rTES-30USM and rTES-120. This study is novel in several ways namely it is the first report of the use of multiple recombinant antigens for serodiagnosis of toxocariasis; the use of rTES-26 (and rTES-32) in Toxocara serodiagnosis; the use of IgG4 assay for rTES-120 and rTES-26 and the use of GST tag in the expression and purification of Toxocara recombinant proteins. These test maybe a significant improvement over commercially available tests for diagnosis of toxocariasis and may be use especially in countries co-endemic with other soil-transmitted helminthes.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/219
dc.language.isoenen_US
dc.subjectBiological Scienceen_US
dc.subjectCloningen_US
dc.subjectRecombinant antigensen_US
dc.subjectToxocariasisen_US
dc.titleCloning, expression and purification of toxocara canis recombinant antigens (rTES-26, rTES-32, rTES-120) and development of serodiagnostic test for toxocariasisen_US
dc.typeThesisen_US
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