Design and evaluation of a recombinant multi-epitope antigen for serodiagnosis of Toxoplasma gondii infection in humans

Abstract

BackgroundSerological investigation remains the primary approach to achieve satisfactory results in Toxoplasma gondii identification. However, the accuracy of the native antigen used in the current diagnostic kits has proven to be insufficient as well as difficult to standardize, so significant efforts have been made to find alternative reagents as capture antigens. Consequently, multi-epitope peptides are promising diagnostic markers, with the potential for improving the accuracy of diagnostic kits. In this study, we described a simple, inexpensive and improved strategy to acquire such diagnostic markers. The study was aimed at producing novel synthetic protein consisting of multiple immunodominant epitopes of several T. gondii antigens.FindingsTo accomplish our goals, a single synthetic gene of approximately 456 bp, which encodes potential epitopes of T. gondii antigens, was successfully constructed using gene assembly PCR. The constructed gene was cloned into a pET32a expression vector and transformed into BL21 E. coli. The entire protein was successfully expressed and purified. Subsequently, the preliminary diagnostic performance of expressed protein was evaluated by developing IgG enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using human sera. The results showed 100 % sensitivity and specificity.ConclusionA purified protein expressing multi-immunodominant epitopes of T. gondii was generated. Further studies are required to evaluate the immunogenicity in animal models and to verify the immuno-reactivity of USM.TOXO1 as a diagnostic antigen.

Evaluation of recombinant multi-epitope proteins for diagnosis of goat schistosomiasis by enzyme-linked immunosorbent assay

“…The other two recombinant multi-epitope proteins with higher sensitivity and specificity in diagnosis of goat schistosomiasis were rBSjPGM-BSj23 (93.4 and 100 %) and rBSjRAD23-2-BSjPGM-BSj23 (89 and 100 %), both also showing lower cross-reactivity with orientobilharziasis (2.7 % for rBSjPGM-BSj23 and 0 % for rBSjRAD23-2-BSjPGM-BSj23) and haemonchosis (0 % for both). The conclusion that constructing multi-epitope antigens can increase the sensitivity of serological detection methods has also been reported in Toxoplasma gondii [ 10 , 11 ]. All above-mentioned show constructing multi-epitope antigens is a worthwhile way.…”

Antigenicity Identification of a Novel Recombinant Multi-Epitope Antigen Based on FlaA and UreB Antigens of Helicobacter pylori

“…Nowadays, the technology of multi-epitope antigens is widespread. Design and construction of multi-epitope DNA and protein antigens can serve as (i) a vaccine candidate against different cancers like breast cancer and cervical cancer (27,28), (ii) a vaccine candidate for the prevention and control of some infectious diseases like Toxoplasmosis, Influenza, and HIV-1 (29)(30)(31), and (iii) an antigen in diagnostic kits for Trypanosoma cruzi, Toxoplasma gondii, Hepatitis C, and Tuberculosis (32)(33)(34)(35). Several virulence genes of H. pylori have been identified, some of which including UreB, HpaA, NapA, FlaA, and FlaB have been investigated as vaccines and serologic diagnostic candidates for H. pylori infection (36)(37)(38)(39).…”

An evaluation of a recombinant multiepitope based antigen for detection of Toxoplasma gondii specific antibodies

“…A single recombinant multiepitope antigen (USM.TOXO1) consisting of nine linear and conserved immunodominant within the SAG1, GRA2 and GRA7 antigens of T. gondii was designed as described previously [ 9 ]. Consequently, the corresponding gene encoding this antigen with final length of 435 bp was constructed by assembly PCR as described by Stemmer (1995) [ 23 ].…”

“…Despite the satisfactory results obtained from the serodiagnosis specifically ELISA, development of standard and reliable reagents remains laborious and expensive [ 7 , 8 ]. Furthermore, the insufficient accuracy of several serodiagnostic tests necessitates the exploration of alternative reagents to be used for diagnostic purposes in the progress of toxoplasmosis control [ 8 , 9 ]. On the basis of this, suggestions were put forward to identify possible future directions of research on the development of accurate diagnostic tests.…”