Designing and Modeling of Multi-epitope Proteins for Diagnosis of Toxocara canis Infection

  • Maryam Ebrahimi
  • Seyyed Javad SeyyedtabaeiEmail author
  • Mohammad Mehdi Ranjbar
  • Farid Tahvildar-biderouni
  • Amirreza Javadi Mamaghani


Serological investigation is the main method to achieve satisfactory results in Toxocara canis diagnosis. The accuracy of the native antigen used in the current diagnostic kits has proven to be insufficient as well as difficult to standardize. Therefore significant efforts have been made to find alternative reagents as capture antigens. Multi-epitope peptides are potential diagnostic markers to improve the accuracy of diagnostic kits. The main aim of this study is the prediction and design of a novel synthetic protein consisting of multiple immunodominant B cell epitopes by Use of three proteins TES-120, TES-30, and TES-26 of T. canis. Primary, secondary and tertiary structures of the proteins were analyzed by using several various online software (ExPasy, IEDB, ABCpred, SVMTriP). Then, B cell construct was assessed by machine learning and Physico-chemical approaches and finally, homology modeling of 3D structure of protein was evaluated. The results of in silico analyses indicated that regions with high immunogenicity for TES-120 protein are located at between residues 97–167, for TES-30 protein are in the residues 52–102, 172–207 and for TES-26 are in the residues 33–83, 130–180. These regions could have good potential features for designing the Multi-epitopes. Finally, selected epitopes were linked to each other by linkers. The average length of the constructs was 342 bp. Also, the high proportion of random coils and extended strands in construct suggest that the protein form antigenic epitopes. Expasy ProtParam classified the constructs with moderate stability and 56.2% residues of constructs were located in favored regions of the Ramachandran plot. In conclusion, immunoinformatics analysis indicated that this multi-epitope peptide can laid a theoretical basis to develop an appropriate diagnostic kit for human toxocariasis.


Multi-epitope Synthetic gene Toxocara canis Bioinformatics softwar 


Compliance with Ethical Standards

Conflict interest

All authors declare that they have no conflict interest.

Supplementary material

10989_2019_9940_MOESM1_ESM.jpg (231 kb)
Electronic supplementary material 1—Transmembrane structure prediction of (A) TES-120 (B) TES-30, (C) TES-26 proteins by TMHMM Server. (JPG 231 kb)
10989_2019_9940_MOESM2_ESM.jpg (168 kb)
Electronic supplementary material 2—Signal peptide prediction of TES proteins using SignalP software: (A) TES-120, (B) TES-30 and (C) TES (JPG 168 kb)
Electronic supplementary material 3—The amino acid sequence of B cell construct, each epitope is separated by a flexible linker (bold). a histidine tag(H6x) is present at the C-terminal end. (PNG 61 kb)


  1. Aghaei S, Riahi SM, Rostami A, Mohammadzadeh I, Javanian M, Tohidi E et al (2018) Toxocara spp. infection and risk of childhood asthma: a systematic review and meta-analysis. Acta Trop 182:298–304PubMedCrossRefGoogle Scholar
  2. Aghamolaie S, Seyyedtabaei SJ, Behniafar H, Foroutan M, Saber V, Hanifehpur H et al (2018) Seroepidemiology, modifiable risk factors and clinical symptoms of Toxocara spp. infection in Northern Iran. Trans R Soc Trop Med Hyg 113:116–122CrossRefGoogle Scholar
  3. Baneth G, Thamsborg SM, Otranto D, Guillot J, Blaga R, Deplazes P et al (2016) Major parasitic zoonoses associated with dogs and cats in Europe. J Comp Pathol 155(1 Suppl 1):S54–S74PubMedCrossRefGoogle Scholar
  4. Besse F, Ephrussi A (2008) Translational control of localized mRNAs: restricting protein synthesis in space and time. Nat Rev Mol Cell Biol 9(12):971–980PubMedCrossRefGoogle Scholar
  5. Bhattacharya A, Tejero R, Montelione GT (2007) Evaluating protein structures determined by structural genomics consortia. Proteins 66(4):778–795PubMedCrossRefGoogle Scholar
  6. Dai JF, Jiang M, Qu LL, Sun L, Wang YY, Gong LL et al (2013) Toxoplasma gondii: enzyme-linked immunosorbent assay based on a recombinant multi-epitope peptide for distinguishing recent from past infection in human sera. Exp Parasitol 133(1):95–100PubMedCrossRefGoogle Scholar
  7. DeLano WL (2002) The PyMOL molecular graphics system. San Carlos, DeLano Scientific, p 700Google Scholar
  8. Despommier D (2005) Toxocariasis: clinical aspects, epidemiological, medical ecology and molecular aspects. Clin Microbiol Rev 16:265–272CrossRefGoogle Scholar
  9. Dipti CA, Jain SK, Navin K (2006) A novel recombinant multiepitope protein as a hepatitis C diagnostic intermediate of high sensitivity and specificity. Protein Expr Purif 47(1):319–328PubMedCrossRefGoogle Scholar
  10. Dorosti H, Eslami M, Negahdaripour M, Ghoshoon MB, Gholami A, Heidari R et al (2019) Vaccinomics approach for developing multi-epitope peptide pneumococcal vaccine. J Biomol Struct Dyn 37(13):3524–3535PubMedCrossRefGoogle Scholar
  11. Fakhri Y, Gasser R, Rostami A, Fan C, Ghasemi S, Javanian M et al (2018) Toxocara eggs in public places worldwide—a systematic review and meta-analysis. Environ Pollut 242:1467–1475PubMedCrossRefGoogle Scholar
  12. Fong MY, Lau YL (2004) Recombinant expression of the larval excretory-secretory antigen TES-120 of Toxocara canis in the methylotrophic yeast Pichia pastoris. Parasitol Res 92(2):173–176PubMedCrossRefGoogle Scholar
  13. Fong MY, Lau YL, Init I, Jamaiah I, Anuar AK, Rahmah N (2003) Recombinant expression of Toxocara canis excretory-secretory antigen TES-120 in Escherichia coli. Southeast Asian J Trop Med Public Health 34(4):723–726PubMedGoogle Scholar
  14. Frank SA (2002) Immunology and evolution of infectious disease. Princeton, Princeton University PressGoogle Scholar
  15. Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31(13):3784–3788PubMedPubMedCentralCrossRefGoogle Scholar
  16. Geourjon C, Deleage G (1995) SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 11(6):681–684PubMedGoogle Scholar
  17. Hajissa K, Zakaria R, Suppian R, Mohamed Z (2015) Design and evaluation of a recombinant multi-epitope antigen for serodiagnosis of Toxoplasma gondii infection in humans. Parasit Vectors 8:315PubMedPubMedCentralCrossRefGoogle Scholar
  18. Kalwy S, Rance J, Young R (2006) Toward more efficient protein expression: keep the message simple. Mol Biotechnol 34(2):151–156PubMedCrossRefGoogle Scholar
  19. Karpenko LI, Bazhan SI, Antonets DV, Belyakov IM (2014) Novel approaches in polyepitope T-cell vaccine development against HIV-1. Expert Rev Vaccines 13(1):155–173PubMedCrossRefGoogle Scholar
  20. Kolaskar AS, Tongaonkar PC (1990) A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 276(1–2):172–174PubMedCrossRefGoogle Scholar
  21. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305(3):567–580PubMedCrossRefGoogle Scholar
  22. Kulkarni R, Sapkal G, Mahishi L, Shil P, Gore MM (2012) Design and characterization of polytope construct with multiple B and TH epitopes of Japanese encephalitis virus. Virus Res 166(1–2):77–86PubMedCrossRefGoogle Scholar
  23. Lv C, Hong Y, Fu Z, Lu K, Cao X, Wang T et al (2016) Evaluation of recombinant multi-epitope proteins for diagnosis of goat schistosomiasis by enzyme-linked immunosorbent assay. Parasit Vectors 9:135PubMedPubMedCentralCrossRefGoogle Scholar
  24. Maizels RM, de Savigny D, Ogilvie BM (1984) Characterization of surface and excretory-secretory antigens of Toxocara canis infective larvae. Parasit Immunol 6(1):23–37CrossRefGoogle Scholar
  25. Maizels RM, Kennedy MW, Meghji M, Robertson BD, Smith HV (1987) Shared carbohydrate epitopes on distinct surface and secreted antigens of the parasitic nematode Toxocara canis. J Immunol 139(1):207–214PubMedGoogle Scholar
  26. Mohamad S, Azmi NC, Noordin R (2009) Development and evaluation of a sensitive and specific assay for diagnosis of human toxocariasis by use of three recombinant antigens (TES-26, TES-30USM, and TES-120). J Clin Microbiol 47(6):1712–1717PubMedPubMedCentralCrossRefGoogle Scholar
  27. Mohammadzadeh I, Riahi SM, Saber V, Darvish S, Amrovani M, Arefkhah N et al (2018) The relationship between Toxocara species seropositivity and allergic skin disorders: a systematic review and meta-analysis. Trans R Soc Trop Med Hyg 112(12):529–537PubMedGoogle Scholar
  28. Noordin R, Smith HV, Mohamad S, Maizels RM, Fong MY (2005) Comparison of IgG-ELISA and IgG4-ELISA for Toxocara serodiagnosis. Acta Trop 93(1):57–62PubMedCrossRefGoogle Scholar
  29. Norhaida A, Suharni M, Liza Sharmini AT, Tuda J, Rahmah N (2008) rTES-30USM: cloning via assembly PCR, expression, and evaluation of usefulness in the detection of toxocariasis. Ann Trop Med Parasitol 102(2):151–160PubMedCrossRefGoogle Scholar
  30. Overgaauw PA (1997) Aspects of Toxocara epidemiology: human toxocarosis. Crit Rev Microbiol 23(3):215–231PubMedCrossRefGoogle Scholar
  31. Pruess M, Apweiler R (2003) Bioinformatics Resources for in silico proteome analysis. J Biomed Biotechnol 2003(4):231–236PubMedPubMedCentralCrossRefGoogle Scholar
  32. Ramakrishna L, Anand KK, Mohankumar KM, Ranga U (2004) Codon optimization of the tat antigen of human immunodeficiency virus type 1 generates strong immune responses in mice following genetic immunization. J Virol 78(17):9174–9189 Epub 2004/08/17 PubMedPubMedCentralCrossRefGoogle Scholar
  33. Roldan WH, Espinoza YA (2009) Evaluation of an enzyme-linked immunoelectrotransfer blot test for the confirmatory serodiagnosis of human toxocariasis. Mem Inst Oswaldo Cruz 104(3):411–418PubMedCrossRefGoogle Scholar
  34. Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5(4):725–738PubMedPubMedCentralCrossRefGoogle Scholar
  35. Rubinsky-Elefant G, Hirata CE, Yamamoto JH, Ferreira MU (2010) Human toxocariasis: diagnosis, worldwide seroprevalences and clinical expression of the systemic and ocular forms. Ann Trop Med Parasitol 104(1):3–23PubMedCrossRefGoogle Scholar
  36. Saadi M, Karkhah A, Nouri HR (2017) Development of a multi-epitope peptide vaccine inducing robust T cell responses against brucellosis using immunoinformatics based approaches. Infect Genet Evol 51:227–234PubMedCrossRefGoogle Scholar
  37. Saha S, Bhasin M, Raghava GP (2005) Bcipep: a database of B-cell epitopes. BMC Genom 6:79CrossRefGoogle Scholar
  38. Sandhu KS, Pandey S, Maiti S, Pillai B (2008) GASCO: genetic algorithm simulation for codon optimization. In Silico Biol 8(2):187–192PubMedGoogle Scholar
  39. Sela-Culang I, Kunik V, Ofran Y (2013) The structural basis of antibody-antigen recognition. Front Immunol 4:302PubMedPubMedCentralCrossRefGoogle Scholar
  40. Singh H, Ansari HR, Raghava GP (2013) Improved method for linear B-cell epitope prediction using antigen's primary sequence. PLoS ONE 8(5):e62216PubMedPubMedCentralCrossRefGoogle Scholar
  41. Siyadatpanah A, Tabatabaei F, Zeydi AE, Spotin A, Fallah-Omrani V, Assadi M et al (2013) Parasitic contamination of raw vegetables in Amol North of Iran. Arch Clin Infect Dis 8(2):e15983CrossRefGoogle Scholar
  42. Smith H, Holland C, Taylor M, Magnaval JF, Schantz P, Maizels R (2009) How common is human toxocariasis? Towards standardizing our knowledge. Trends Parasitol 25(4):182–188PubMedCrossRefGoogle Scholar
  43. Wiederstein M, Sippl MJ (2007) ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res 35:W407–W410CrossRefGoogle Scholar
  44. Yamasaki H, Araki K, Lim PK, Zasmy N, Mak JW, Taib R et al (2000) Development of a highly specific recombinant Toxocara canis second-stage larva excretory-secretory antigen for immunodiagnosis of human toxocariasis. J Clin Microbiol 38(4):1409–1413PubMedPubMedCentralGoogle Scholar
  45. Yang ZR (2004) Biological applications of support vector machines. Brief Bioinform 5(4):328–338PubMedCrossRefGoogle Scholar
  46. Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y (2015) The I-TASSER suite: protein structure and function prediction. Nat Methods 12(1):7–8PubMedPubMedCentralCrossRefGoogle Scholar
  47. Zhang W, Xiong Y, Zhao M, Zou H, Ye X, Liu J (2011) Prediction of conformational B-cell epitopes from 3D structures by random forests with a distance-based feature. BMC Bioinform 12:341CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019
corrected publication 2019

Authors and Affiliations

  • Maryam Ebrahimi
    • 1
  • Seyyed Javad Seyyedtabaei
    • 2
    Email author
  • Mohammad Mehdi Ranjbar
    • 3
  • Farid Tahvildar-biderouni
    • 2
  • Amirreza Javadi Mamaghani
    • 2
  1. 1.Department of Medical Parasitology and Mycology, School of MedicineStudent Research Committee, Shahid Beheshti University of Medical SciencesTehranIran
  2. 2.Department of Medical Parasitology and Mycology, School of MedicineShahid Beheshti University of Medical SciencesTehranIran
  3. 3.Department of ImmunologyRazi Vaccine and Sera Research InstuteKarajIran

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