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Licensed Unlicensed Requires Authentication Published by De Gruyter July 5, 2017

Structuraland antigenic analysis of a new Rhoptry Pseudokinase Gene (ROP54) in Toxoplasma gondii

  • Jian Zhou , Gang Lu , Lin Wang , Aihua H. Zhou , Yali L. Han , Jingjing J. Guo , Pengxia X. Song , Huaiyu Y. Zhou , Hua Cong , Ming Hou and Shenyi Y. He EMAIL logo
From the journal Acta Parasitologica


Toxoplasma gondii is defined as an obligate intracellular apicomplexan parasite and influences approximatelyone-third of the human all over the world. ROP54 protein is expressed in the rhoptry of Toxoplasma gondii. In the present study, we used SMART software to analyzethe secondary structure of ROP54. The 3D model of ROP54 protein was constructed and analyzed using SWISS-MODEL server and VMD software. The structure results fully showed that ROP54 proteinis an importantmember from the ROP family. Moreover, DNAMAN software and Epitope Database online service were used to analyze liner-B cell epitopes and Th-cell epitopes of the protein. The bioinformatics prediction of ROP54 protein could provide positive information on treatment and vaccine for toxoplasmosis. Furthermore, ROP54 gene was obtained from PCR, and a recombinant eukaryotic expression vector (pEGFP-ROP54) was constructed in the following study. After identification of enzyme digestion, the constructed plasmid was transfected into HEK 293-T cells. The RT-PCR result suggested that the recombinant plasmid could transcribe successfully in HEK 293-T cell.


This work was supported, in part, by grants from the National Natural Science Foundation of China (Grant Nos. 81071373 and 81271857), the State Key Laboratory of Veterinary Etiological Biology (Grant No. SKLVEB2011KFKT005) and the Shandong Provincial Natural Science Foundation (Grant No. ZR2009CM079).


Alaganan A., Fentress S.J., Tang K., Wang Q., Sibley L.D. 2014. Toxoplasma GRA7 effector increases turnover of immunity-related GTPases and contributes to acute virulence in the mouse. Proceedings of the National Academy of Sciences USA, 111, 1126–1131. 10.1073/pnas.1313501111Search in Google Scholar

Bai Y., He S., Zhao G.H., Chen L., Shi N., Zhou H.Y., et al. 2012. Toxoplasma gondii: bioinformatics analysis, cloning and expression of a novel protein TgIMP1. Experimental Parasitology, 132, 458–464. 10.1016/j.exppara.2012.09.015Search in Google Scholar

Behnke M.S., Fentress S.J., Mashayekhi M., Li L.X., Taylor G.A., Sibley L.D. 2012. The polymorphic pseudokinase ROP5 controls virulence in Toxoplasma gondii by regulating the active kinase ROP18. PLoS Pathogens, 8, e1002992. 10.1371/journal.ppat.1002992Search in Google Scholar

Boothroyd J.C., Grigg M.E. 2002. Population biology of Toxoplasma gondii and its relevance to human infection: do different strains cause different disease? Current Opinion in Microbiology, 5, 438–442. 10.1016/S1369-5274(02)00349-1Search in Google Scholar

Bradley P.J., Ward C., Cheng S.J., Alexander D.L., Coller S., Coombs G.H., et al. 2005. Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii. Journal of Biological Chemistry, 280, 34245–34258. 10.1074/jbc.M504158200Search in Google Scholar PubMed

Butcher B.A., Fox B.A., Rommereim L.M., Kim S.G., Maurer K.J., Yarovinsky F., et al. 2011. Toxoplasma gondii rhoptry kinase ROP16 activates STAT3 and STAT6 resulting in cytokine inhibition and arginase-1-dependent growth control. PLoS Pathogens, 7, e1002236. 10.1371/journal.ppat.1002236Search in Google Scholar PubMed PubMed Central

Carter J.M., Loomis-Price L. 2004. B cell epitope mapping using synthetic peptides. Current Protocols in Immunology, 10.1002/0471142735Search in Google Scholar

Cong H., Gu Q.M., Yin H.E., Wang J.W., Zhao Q.L., Zhou H.Y., et al.2008. Multi-epitope DNA vaccine linked to the A2/B subunit of cholera toxin protect mice against Toxoplasma gondii. Vaccine, 26, 3913–3921. 10.1016/j.vaccine.2008.04.046Search in Google Scholar PubMed

Constant S., Pfeiffer C., Woodard A., Pasqualini T., Bottomly K. 1995. Extent of T cell receptor ligation can determine the functional differentiation of native CD4+ T cells. The Journal of Experimental Medicine, 182, 1591–1596. 10.1084/jem.182.5.1591Search in Google Scholar PubMed PubMed Central

Elliot W. Kim., Santhosh M. Nadipuram., Ashley L. Tetlow., William D. Barshop., et al. 2016. The Rhoptry Pseudokinase ROP54 Modulates Toxoplasma gondii Virulence and Host GBP2 Loading. Msphere, 1, e00045–16. 10.1128/mSphere.00045-16Search in Google Scholar PubMed PubMed Central

Ferra B., Holec-Gąsior L., Kur J. 2015. A new Toxoplasma gondiichimeric antigen containing fragments of SAG2, GRA1, andimpact of immunodominant sequences size on its diagnostic usefulness. Parasitology Research, 114, 3291–3299. 10.1007/s00436-015-4552-6Search in Google Scholar

Gao J., Faraggi E., Zhou Y., Ruan J., Kurgan L. 2012. BEST: improved prediction of B-cell epitopes from antigen sequences. PLoS One, 7, e40104 p. 10.1371/journal.pone.0040104Search in Google Scholar

Gershoni JM., Stern B., Denisova G.1997. Combinatorial libraries, epitope structure and the prediction of protein conformations. Immunology Today, 18, 108–110. 10.1016/S0167-5699(97)01024-4Search in Google Scholar

Guex N., Peitsch MC., Schwede T. 2009. Automated comparative protein structure modeling with SWISS-MODEL and Swiss- Pdb Viewer: a historical perspective. Electrophoresis, 30, S162–S173. 10.1002/elps.200900140Search in Google Scholar

Guex N., Peitsch N.C. 1997. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling.Electrophoresis, 18, 2714–2723. 10.1002/elps.1150181505Search in Google Scholar

Hakansson S., Charron A.J., Sibley L.D. 2001. Toxoplasma evacuoles: atwo-step process of secretion and fusion forms the parasitophorous vacuole. The EMBO Journal, 20, 3132–3144. 10.1093/emboj/20.12.3132Search in Google Scholar

Hwang S., Khan I.A.2015. CD8+ T cell immunity in an encephalitis model of Toxoplasma gondii infection. SEMINARS IN IMMUNOPATHOLOGY, 37, 271–279. 10.1007/s00281-015-0483-7Search in Google Scholar

Kopp J., Schwede T. 2006. The SWISS-MODEL Repository: new features and functionalities. Nucleic Acids Research, 34, 315–318. 10.1093/nar/gkj056Search in Google Scholar

Kyte J., Doolittle R.F. 1982. A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology, 157, 105–132. 10.1016/0022-2836(82)90515-0Search in Google Scholar

Liu K.Y., Zhang D.B., Wei Q.K., Li J., Li G.P., Yu J.Z. 2006. Biological role of surface Toxoplasma gondii antigen in development of vaccine. World Journal of Gastroenterology, 12, 2363–2368. 10.3748/WJG.v12.i15.2363Search in Google Scholar

Nielsen H.V., Lauemoller S.L., Christiansen L., Buus S., Fomsgaard A., Petersen E. 1999. Complete protection against lethal Toxoplasma gondii infection in mice immunized with a plasmid encoding the SAG1 gene. Infection and Immunity, 67, 6358–6363. 10.1016/S0960-9822(99)80167-5Search in Google Scholar

Ong Y.C., Reese M.L., Boothroyd J.C. 2010. Toxoplasma rhoptry protein16 (ROP16) subverts host function by direct tyrosine phosphorylation of STAT6. Journl of Biological Chemistry, 285, 28731–28740. 10.1074/jbc.M110.112359Search in Google Scholar

Romano P., Giugno R., Pulvirenti A. 2011. Tools and collaborative environments for bioinformatics research. Briefings in Bioinformatics, 12, 549–561. 10.1093/bib/bbr055Search in Google Scholar

Siachoque H., Guzman F., Burgos J., Patarroyo M.E., Gomez Marin J.E. 2006. Toxoplasma gondii: immunogenicity and protection by P30 peptides in a murine model. Experimental Parasitology, 114, 62–65. 10.1016/j.exppara.2006.02.005Search in Google Scholar

Subramani A., Floudas C.A. 2012. Structure prediction of loops with fixed and flexible stems. The Journal of Physical Chemistry B, 116, 6670–6682.10.1021/jp2113957Search in Google Scholar

Tong J.C., Tammi M.T. 2008. Prediction of protein allergenicity using local description of amino acid sequence. Frontiers in Bioscience Landmark, 13, 6072–6087. org/10.2741/3138Search in Google Scholar

Van Regenmortel MH. 2009. What is a B-cell epitope?. Methods in Molecular Biology, 524, 3–20. 10.1007/978-1-59745-450-6_1Search in Google Scholar

Wang Y.H., Wang G.X., Ou J.T., Yin H., Zhang D.L. 2014. Analyzing and identifying novel B cell epitopes within Toxoplasma gondii GRA4. Parasites & Vectors, 7, 474. in Google Scholar

Wang Y.H., Wang M., Wang G.X., Pang A.N., Fu B.Q., Yin H., et al.2011. Increasedsurvival time in mice vaccinated with a branched lysine multipleantigenic peptide containing B- and T-cell epitopes from T. gondii antigens. Vaccine, 29, 8619–8623. 10.1016/j.vaccine.2011.09.016Search in Google Scholar

Welling G.W., Weijer W.J., Van der Zee R., Welling-Wester S. 1985. Prediction of sequential antigenic regions in proteins. FEBS Letters, 188, 215–218. 10.1016/0014-5793(85)80374-4Search in Google Scholar

Yamamoto M., Standley D.M., Takashima S., Saiga H., Okuyama M., Kayama H., et al. 2009. A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation ofStat3. Journal of Experimental Medicine, 206, 2747–2760. 10.1084/jem.20091703Search in Google Scholar PubMed PubMed Central

Yuan Z.G., Zhang X.X., He X.H., Petersen E., Zhou D.H., He Y., et al.2011. Protective immunity induced by Toxoplasma gondii rhoptry protein 16 against toxoplasmosis in mice. Clinical and Vaccine Immunology, 18, 119–124. 10.1128/CVI.00312-10Search in Google Scholar PubMed PubMed Central

Yuan Z.G., Zhang X.X., Lin R.Q., Petersen E., He S., Yu M., et al.2011. Protective effect against toxoplasmosis in mice induced by DNA immunization with gene encoding Toxoplasma gondii ROP18. Vaccine, 29, 6614–6619. 10.1016/j.vaccine.2011.06.110Search in Google Scholar PubMed

Zhang T.E., Yin L.T., Li R.H., Wang H.L., Meng X.L., Yin G.R. 2015. Protective immunity induced by peptides ofAMA1, RON2 and RON4 containing T-and B-cellepitopes via an intranasal route against toxoplasmosis in mice. Parasites & Vectors, 8, 15. 10.1186/s13071-015-0636-5Search in Google Scholar PubMed PubMed Central

Received: 2016-10-19
Revised: 2017-3-10
Accepted: 2017-3-27
Published Online: 2017-7-5
Published in Print: 2017-9-26

© 2017 W. Stefański Institute of Parasitology, PAS

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