Cloning of 1183 bp Fragment from Rhoptry Protein I (ROPI) Gene of Toxoplasma gondii (RH) in Expression Prokaryote Plasmid PET32a

authors:

avatar Zahra Eslamirad 1 , * , avatar Behzad Ghorbanzadeh 1 , avatar Reza Hajihossein 1 , avatar Hamid Abtahi 2 , avatar Mahdi Mosayebi 1 , avatar Saeedeh Shojaee 3 , avatar Abdolrahim Sadeghi 4

Department of Parasitology, Arak University of Medical Sciences, Arak, Iran
Department of Microbiology, Arak University of Medical Sciences, Arak, Iran
Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Department of Biochemistry, Arak University of Medical Sciences, Arak, Iran
Zahedan Journal of Research in Medical Sciences: Vol.15, issue 10; e92828
published online: May 12, 2013
article type: Research Article
received: January 21, 2012
revised: March 14, 2012
accepted: April 23, 2012

how to cite: Eslamirad Z, Ghorbanzadeh B, Hajihossein R, Abtahi H, Mosayebi M, et al. Cloning of 1183 bp Fragment from Rhoptry Protein I (ROPI) Gene of Toxoplasma gondii (RH) in Expression Prokaryote Plasmid PET32a. Zahedan J Res Med Sci. 2013;15(10):e92828. 

Abstract

Background : Toxoplasma gondii is an obligatory intracellular protozoan. Considering to high prevalence of this disease the best way to reduce the raised loses is prevention of human and animal infection, rapid diagnosis, differentiation between acute and chronic disease. Rhoptry protein 1 of Toxoplasma gondii is an excretory-secretory antigen that exists in the most stages of life cycle. According to specifications of excretory-secretory antigen that seems this antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit. The main object of the present work was cloning rhoptry protein 1 (ROP1) gene of Toxoplasma gondii (RH) in a cloning vector for further production of rhoptry proteins.
Materials and Methods : Genomic DNA was extracted by phenol-chloroform method. The ROP1 fragment was amplified by PCR. This product was approved by sequencing and was cloned between the EcoR1 and Sal1 sites of the pTZ57R/T vector. Then transformed into Escherichia coli DH5α strain and screened by IPTG and X-Gal. After isolating of this gene from pTZ57R/T, it was subcloned into pET32a plasmid.
Results : The plasmid was purified and approved by electrophoresis, enzyme restriction and PCR. After isolating of this gene from pTZ57R/T, it was subcloned into pET32a plasmid. After enzyme restriction and electrophoresis a fragment about 1183bp was separated from pET32a.
Conclusion : Recombinant plasmid of ROP1 gene was constructed and ready for future study. That seems the antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit.

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