Molecular Cloning, Expression and Characterization of Plasmid Encoding Rhomboid 4 (ROM4) of Tachyzoite of Toxoplasma gondii RH Strain
-
Mohammad Taghi RAHIMI
,
Shahabeddin SARVI
,
Mahdi SHARIF
,
Saeid ABEDIANKENARI
,
Ehsan AHMADPOUR
,
Reza VALADAN
,
Ahmad DARYANI
Abstract
Background: The objective of this study was to clone, express and characterize the gene encoding rhomboid 4 (ROM4) proteins, a vital gene in surface adhesion and host cell invasion process of tachyzoite of T. gondii in an appropriate expression vector and eukaryotic cell for production of recombinant protein.
Methods: Toxoplasma RNA was isolated from tachyzoites (RH strain) and complementary DNA was synthesized. Oligonucleotide primer pair was designed based on Toxoplasma ROM4 gene sequence with XhoI and EcoRI restriction sites at 5´ end of forward and reverse primers, respectively. ROM4 gene was amplified by PCR, cloned into pTG19-T vector and the recombinant plasmid was sequenced. The gene was subcloned into pcDNA3 plasmid and expressed in CHO cells as eukaryotic cell. SDS-PAGE and western blotting were performed for protein determination and verification.
Results: Cloning of ROM4 gene in pTG19-T vector was confirmed by colony-PCR and enzymatic digestion. The results of enzymatic digestion and gene sequencing confirmed successful cloning and subcloning procedures. The nucleotide sequence of the cloned ROM4 gene showed 99% homology compared to the corresponding sequences of original gene. SDS-PAGE and western blotting analyses of the purified protein revealed a single band having expected size of 65 kDa.
Conclusion: This eukaryotic expression system is an appropriate system for high-level recombinant protein production of ROM4 gene from T. gondii tachyzoites used as antigenic component for serological assay and vaccine development.Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol. 2000; 30(12-13):1217-58.
Rahimi MT, Mahdavi SA, Javadian B et al. High seroprevalence of Toxoplasma gondii antibody in HIV/AIDS individuals from North of Iran. Iran J Parasitol. 2015; 10(4):584-9.
Sarvi S, Daryani A, Rahimi MT et al. Cattle toxoplasmosis in Iran: a systematic review and meta–analysis. Asian Pac J Trop Med. 2015; 8(2):120-6.
Kazemi B, Bandehpour M, Maghen L et al. Gene cloning of 30 kDa Toxoplasma gondii tachyzoites surface antigen (SAG1). Iran J Parasitol. 2007; 2(2): 1-8.
Dubey JP, Jones JL. Toxoplasma gondii infection in humans and animals in the United States. Int J Parasitol. 2008; 38(11):1257-78.
Tekkesin N. Diagnosis of toxoplasmosis in pregnancy: a review. HOAJ Biol. 2012; 1(1): 9-12.
Rahimi MT, Daryani A, Sarvi S et al. Cats and Toxoplasma gondii: A systematic review and meta-analysis in Iran. Onderstepoort J Vet Res. 2015;82(1):823.
Chen J, Li ZY, Petersen E et al. DNA vaccination with genes encoding Toxoplasma gondii antigens ROP5 and GRA15 induces protective immunity against toxoplasmosis in Kunming mice. Expert Rev Vaccines. 2015;14(4):617-24.
Santos JM, Ferguson DJ, Blackman MJ et al. Intra membrane cleavage of AMA1 triggers Toxoplasma to switch from an invasive to a replicative mode. Science. 2011;331(6016):473-7.
Zhang NZ, Xu Y, Wang M et al. Protective efficacy of two novel DNA vaccines expressing Toxoplasma gondii rhomboid 4 and rhomboid 5 proteins against acute and chronic toxoplasmosis in mice. Expert Rev Vaccines. 2015;14 (9):1289-97.
Abdi J, Kazemi B, Mohebali M et al. Gene cloning, expression and serological evaluation of the 12-kDa antigen-B subunit from Echinococcus granulosus. Ann Trop Med Parasitol. 2010; 104(5):399-407.
Zhang NZ, Chen J, Wang M et al. Vaccines against Toxoplasma gondii: new developments and perspectives. Expert Rev Vaccines. 2013;12(11):1287-99.
Buxton D, Thomson K, Maley S et al. Vaccination of sheep with a live incomplete strain (S48) of Toxoplasma gondii and their immunity to challenge when pregnant. Vet Rec. 1991;129(5):89-93.
Dowse TJ, Pascall JC, Brown KD et al. Apicomplexan rhomboids have a potential role in microneme protein cleavage during host cell invasion. Int J Parasitol. 2005;35(7):747-56.
Sibley LD. The roles of intramembrane proteases in protozoan parasites. Biochim Biophys Acta. 2013;1828(12):2908-15.
Brossier F, Jewett TJ, Sibley LD et al. A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma. Proc Natl Acad Sci U S A. 2005; 102(11):4146-51.
Li J, Han Q, Gong P et al. Toxoplasma gondii rhomboid protein 1 (TgROM1) is a potential vaccine candidate against toxoplasmosis. Vet Parasitol. 2012:184(2-4):154-60.
Shen B, Buguliskis JS, Lee TD et al. Functional analysis of rhomboid proteases during Toxoplasma invasion. MBio. 2014; 5(5):e01795-14.
Huynh MH, Carruthers VB. Toxoplasma MIC2 is a major determinant of invasion and virulence. PLoS Pathog. 2006;2(8):e84.
Yang HL, Xiao JH, Liang Y et al. Cloning, sequencing and expressing of microneme protein 1 partial gene in Toxoplasma gondii ZS2 isolate. Zhonghua Yu Fang Yi Xue Za Zhi. 2003;37(1):29-32.
Artama WT, Dewi NNA, Subekti DT. Cloning gene encoding micronema 3 (Mic3) protein of tachyzoite Toxoplasma gondii local isolate. Indonesian J Biotechnol. 2011; 10(1): 5-9.
Babaie J, Zare M, Sadeghiani G et al. Bacterial production of dense granule antigen GRA8 of Toxoplasma gondii. Iran Biomed J. 2009:13(3):145-51.
Naserifar R, Ghaffarifar F, Dalimi et al. Cloning of Toxoplasma Gondii Granular Antigen 5(GRA5) in Expression Eukaryotic Plasmid pcDNA3 and its Expression on CHO CellJ Ilam Uni Med SCI. 2011; 19 (3): 1-12.
| Files | ||
| Issue | Vol 12 No 4 (2017) | |
| Section | Original Article(s) | |
| Keywords | ||
| Apicomplexa Toxoplasma gondi Rhomboid 4 Cloning Expression | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
