- Introduction
- Materials and Methods
- Generation of ROP2 synthetic gene.
- Plasmid construction and transformation.
- Expression of recombinant protein.
- Protein harvesting from buffered complex medium containing methanol.
- Purification of recombinant ROP2.
- Gel electrophoresis and Western blotting.
- Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.
- Sensitivity and specificity of recombinant ROP2.
- Results
- Discussion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Singh S, 2003. Mother-to-child transmission and diagnosis of Toxoplasma gondii infection during pregnancy. Indian J Med Microbiol 21: 69–76.
-
2.
Hill D, Dubey JP, 2002. Toxoplasma gondii: transmission, diagnosis and prevention. Clin Microbiol Infect 8: 634–640.
-
3.
Rorman E, Zamir CS, Rilkis I, Ben-David H, 2006. Congenital toxoplasmosis: prenatal aspects of Toxoplasma gondii infection. Reprod Toxicol 21: 458–472.
-
4.
Alford CA Jr, Stagno S, Reynolds DW, 1974. Congenital toxoplasmosis: clinical, laboratory, and therapeutic considerations, with special reference to subclinical disease. Bull N Y Acad Med 50: 160–181.
-
5.
Fatoohi AF, Cozon GJ, Greenland T, Ferrandiz J, Bienvenu J, Picot S, Peyron F, 2002. Cellular immune responses to recombinant antigens in pregnant women chronically infected with Toxoplasma gondii. Clin Diagn Lab Immunol 9: 704–707.
-
6.
Wong S-Y, Remington JS, 1993. Biology of Toxoplasma gondii. AIDS 7: 299–316.
-
7.
Beckers CJ, Dubremetz JF, Mercereau-Puijalon O, Joiner KA, 1994. The Toxoplasma gondii rhoptry protein Rop-2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host-cell cytoplasm. J Cell Biol 127: 947–961.
-
8.
Saavedra R, Becerril MA, Dubeaux C, Lippens R, De Vos MJ, Herion P, Bollen A, 1996. Epitopes recognized by human T lymphocytes in the ROP2 protein antigen of Toxoplasma gondii. Infect Immun 64: 3858–3862.
-
9.
Martin V, Arcavi M, Santillan G, Amendoeira MR, De Souza Neves E, Griemberg G, Guarnera E, Garberi JC, Angel SO, 1998. Detection of human Toxoplasma-specific immunoglobulins A, M, and G with a recombinant Toxoplasma gondii rop2 protein. Clin Diagn Lab Immunol 5: 627–631.
-
10.
Vercammen M, Scorza T, Huygen K, De Braekeleer J, Diet R, Jacobs D, Saman E, Verschueren H, 2000. DNA vaccination with genes encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice. Infect Immun 68: 38–45.
-
11.
Leyva R, Herion P, Saavedra R, 2001. Genetic immunization with plasmid DNA coding for the ROP2 protein of Toxoplasma gondii. Parasitol Res 87: 70–79.
-
12.
Cregg JM, Vedvick TS, Raschke WC, 1993. Recent advances in the expression of foreign genes in Pichia pastoris. Nat Biotechnol 11: 905–910.
-
13.
Kalidas C, 2004. Pichia pastoris. Encyclopedia of Food Microbiology, San Diego, CA: Academic Press, 1686–1692.
-
14.
Nishimura A, Morita M, Nishimura Y, Sugino Y, 1990. A rapid and highly efficient method for preparation of competent Escherichia coli cells. Nucleic Acids Res 18: 6169.
-
15.
Aubert D, Maine GT, Villena I, Hunt JC, Howard L, Sheu M, Brojanac S, Chovan LE, Nowlan SF, Pinon JM, 2000. Recombinant antigens to detect Toxoplasma gondii-specific immunoglobulin G and immunoglobulin M in human sera by enzyme immunoassay. J Clin Microbiol 38: 1144–1150.
-
16.
Pfrepper KI, Enders G, Gohl M, Krczal D, Hlobil H, Wassenberg D, Soutschek E, 2005. Seroreactivity to and avidity for recombinant antigens in toxoplasmosis. Clin Diagn Lab Immunol 12: 977–982.
-
17.
Vangelder P, Bosman F, Demeuter F, Vanheuverswyn H, Herion P, 1993. Serodiagnosis of toxoplasmosis by using a recombinant form of the 54-kilodalton rhoptry antigen expressed in Escherichia coli. J Clin Microbiol 31: 9–15.
-
18.
Cregg JM, Barringer KJ, Hessler AY, Madden KR, 1985. Pichia pastoris as a host system for transformations. Mol Cell Biol 5: 3376–3385.
-
19.
Chiruvolu V, Cregg JM, Meagher MM, 1997. Recombinant protein production in an alcohol oxidase-defective strain of Pichia pastoris in fedbatch fermentations. Enzyme Microb Technol 21: 277–283.
-
20.
Minning S, Serrano A, Ferrer P, Solá C, Schmid RD, Valero F, 2001. Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris. J Biotechnol 86: 59–70.
-
21.
Boettner M, Prinz B, Holz C, Stahl U, Lang C, 2002. High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris. J Biotechnol 99: 51–62.
-
22.
Tang J, Xie QX, Pan SP, Xiao LJ, Dong L, Zhang CX, Sun CJ, 2003. Expression of recombinant human zona pellucida-3 protein (rhZP3) in Pichia pastoris. Sheng Wu Gong Cheng Xue Bao 19: 758–762.
-
23.
Gatkowska J, Dziadek B, Brzostek A, Dziadek J, Dzitko K, Dlugonska H, 2010. Determination of diagnostic value of Toxoplasma gondii recombinant ROP2 and ROP4 antigens in mouse experimental model. Pol J Microbiol 59: 137–141.
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Evaluation of Pichia pastoris–Expressed Recombinant Rhoptry Protein 2 of Toxoplasma gondii for Its Application in Diagnosis of Toxoplasmosis
Phooi Yee Chang
Phooi Yee Chang
Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
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Mun Yik Fong
Mun Yik Fong
Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
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Veeranoot Nissapatorn
Veeranoot Nissapatorn
Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
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Yee Ling Lau
Yee Ling Lau
Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
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Rhoptry protein 2 (ROP2) of Toxoplasma gondii is a rhoptry-secreted protein that plays a critical role in parasitophorous vacuole membrane formation during invasion. In previous studies, ROP2 has been shown to be efficient in triggering humoral and cell-mediated responses. High immunogenicity of ROP2 makes it a potential candidate for diagnosis and vaccination against toxoplasmosis. In this study, the ROP2 gene was cloned into pPICZα A expression vector and extracellularly expressed in the yeast Pichia pastoris, which has numerous advantages over other expression systems for eukaryotic proteins expression. The effectiveness of the secreted recombinant ROP2 as a diagnosis agent was assessed by Western Blot with 200 human serum samples. Recombinant ROP2 reacted with toxoplasmosis-positive human serum samples and yielded an overall sensitivity of 90% and specificity of 95%. However, recombinant ROP2 is a better marker for detection of IgG (91.7%) rather than IgM (80%).
Author Notes
Financial support: This study was supported by University of Malaya Postgraduate Research Grant (PS195-2009C), UMRG (RG039/09HTM), and the High Impact Research Fund (E000013-20001).
Authors' addresses: Phooi Yee Chang, Mun Yik Fong, Veeranoot Nissapatorn, and Yee Ling Lau, Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia, E-mails: phooiyee@gmail.com, fongmy@um.edu.my, veeranoot@um.edu.my, and lauyeeling@um.edu.my.
Reprint requests: Yee Ling Lau, Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia, E-mail: lauyeeling@um.edu.my.
- Introduction
- Materials and Methods
- Generation of ROP2 synthetic gene.
- Plasmid construction and transformation.
- Expression of recombinant protein.
- Protein harvesting from buffered complex medium containing methanol.
- Purification of recombinant ROP2.
- Gel electrophoresis and Western blotting.
- Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.
- Sensitivity and specificity of recombinant ROP2.
- Results
- Discussion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Singh S, 2003. Mother-to-child transmission and diagnosis of Toxoplasma gondii infection during pregnancy. Indian J Med Microbiol 21: 69–76.
-
2.
Hill D, Dubey JP, 2002. Toxoplasma gondii: transmission, diagnosis and prevention. Clin Microbiol Infect 8: 634–640.
-
3.
Rorman E, Zamir CS, Rilkis I, Ben-David H, 2006. Congenital toxoplasmosis: prenatal aspects of Toxoplasma gondii infection. Reprod Toxicol 21: 458–472.
-
4.
Alford CA Jr, Stagno S, Reynolds DW, 1974. Congenital toxoplasmosis: clinical, laboratory, and therapeutic considerations, with special reference to subclinical disease. Bull N Y Acad Med 50: 160–181.
-
5.
Fatoohi AF, Cozon GJ, Greenland T, Ferrandiz J, Bienvenu J, Picot S, Peyron F, 2002. Cellular immune responses to recombinant antigens in pregnant women chronically infected with Toxoplasma gondii. Clin Diagn Lab Immunol 9: 704–707.
-
6.
Wong S-Y, Remington JS, 1993. Biology of Toxoplasma gondii. AIDS 7: 299–316.
-
7.
Beckers CJ, Dubremetz JF, Mercereau-Puijalon O, Joiner KA, 1994. The Toxoplasma gondii rhoptry protein Rop-2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host-cell cytoplasm. J Cell Biol 127: 947–961.
-
8.
Saavedra R, Becerril MA, Dubeaux C, Lippens R, De Vos MJ, Herion P, Bollen A, 1996. Epitopes recognized by human T lymphocytes in the ROP2 protein antigen of Toxoplasma gondii. Infect Immun 64: 3858–3862.
-
9.
Martin V, Arcavi M, Santillan G, Amendoeira MR, De Souza Neves E, Griemberg G, Guarnera E, Garberi JC, Angel SO, 1998. Detection of human Toxoplasma-specific immunoglobulins A, M, and G with a recombinant Toxoplasma gondii rop2 protein. Clin Diagn Lab Immunol 5: 627–631.
-
10.
Vercammen M, Scorza T, Huygen K, De Braekeleer J, Diet R, Jacobs D, Saman E, Verschueren H, 2000. DNA vaccination with genes encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice. Infect Immun 68: 38–45.
-
11.
Leyva R, Herion P, Saavedra R, 2001. Genetic immunization with plasmid DNA coding for the ROP2 protein of Toxoplasma gondii. Parasitol Res 87: 70–79.
-
12.
Cregg JM, Vedvick TS, Raschke WC, 1993. Recent advances in the expression of foreign genes in Pichia pastoris. Nat Biotechnol 11: 905–910.
-
13.
Kalidas C, 2004. Pichia pastoris. Encyclopedia of Food Microbiology, San Diego, CA: Academic Press, 1686–1692.
-
14.
Nishimura A, Morita M, Nishimura Y, Sugino Y, 1990. A rapid and highly efficient method for preparation of competent Escherichia coli cells. Nucleic Acids Res 18: 6169.
-
15.
Aubert D, Maine GT, Villena I, Hunt JC, Howard L, Sheu M, Brojanac S, Chovan LE, Nowlan SF, Pinon JM, 2000. Recombinant antigens to detect Toxoplasma gondii-specific immunoglobulin G and immunoglobulin M in human sera by enzyme immunoassay. J Clin Microbiol 38: 1144–1150.
-
16.
Pfrepper KI, Enders G, Gohl M, Krczal D, Hlobil H, Wassenberg D, Soutschek E, 2005. Seroreactivity to and avidity for recombinant antigens in toxoplasmosis. Clin Diagn Lab Immunol 12: 977–982.
-
17.
Vangelder P, Bosman F, Demeuter F, Vanheuverswyn H, Herion P, 1993. Serodiagnosis of toxoplasmosis by using a recombinant form of the 54-kilodalton rhoptry antigen expressed in Escherichia coli. J Clin Microbiol 31: 9–15.
-
18.
Cregg JM, Barringer KJ, Hessler AY, Madden KR, 1985. Pichia pastoris as a host system for transformations. Mol Cell Biol 5: 3376–3385.
-
19.
Chiruvolu V, Cregg JM, Meagher MM, 1997. Recombinant protein production in an alcohol oxidase-defective strain of Pichia pastoris in fedbatch fermentations. Enzyme Microb Technol 21: 277–283.
-
20.
Minning S, Serrano A, Ferrer P, Solá C, Schmid RD, Valero F, 2001. Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris. J Biotechnol 86: 59–70.
-
21.
Boettner M, Prinz B, Holz C, Stahl U, Lang C, 2002. High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris. J Biotechnol 99: 51–62.
-
22.
Tang J, Xie QX, Pan SP, Xiao LJ, Dong L, Zhang CX, Sun CJ, 2003. Expression of recombinant human zona pellucida-3 protein (rhZP3) in Pichia pastoris. Sheng Wu Gong Cheng Xue Bao 19: 758–762.
-
23.
Gatkowska J, Dziadek B, Brzostek A, Dziadek J, Dzitko K, Dlugonska H, 2010. Determination of diagnostic value of Toxoplasma gondii recombinant ROP2 and ROP4 antigens in mouse experimental model. Pol J Microbiol 59: 137–141.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 1561 | 1388 | 26 |
| Full Text Views | 803 | 16 | 1 |
| PDF Downloads | 91 | 13 | 1 |