A preliminary Study: Expression of Rhoptry Protein 1 (ROP1) Toxoplasma gondii in Prokaryote System

Abstract

Background:Toxoplasma gondii is an obligatory intracellular protozoan parasite, which infects human beings. Since the current antigens used for diagnosis or vaccination are contaminated with non parasitic material in which the parasite is grown, it is tried to produce recombinant antigens to design vaccines against toxoplasmosis, or make diagnostic kits. Choosing the type of antigen to produce recombinant vaccine or diagnostic kits is considerably important. The rhoptry protein 1 is one of the excretory-secretary antigens of Toxoplasma which seems to be an appropriate candidate in production of recombinant vaccines and diagnostic kits. Objectives: The current study aimed to produce rhoptry protein 1 from Iranian strains of Toxoplasma for further investigations. Materials and Methods: Genomic DNA was isolated from tachyzoite of parasite by phenol chloroform method and gene fragment was amplified by polymerase chain reaction. The polymerase chain reaction products were ligated into restriction enzymes sites of pTZ57R/T cloning vector. The product was sub-cloned into a prokaryotic expression plasmid (pET32a). The recombinant expression vector containing rhoptry protein 1 sequence was transformed into Escherichia coli BL21 pLysS and was induced by isopropyl β-D-1-thiogalactopyranoside. The sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting methods were used to confirm the production of protein. Results:The result showed that the desired molecular weight protein is produced. Recombinant protein was confirmed by western blot using Ni-NTA-conjugate. Conclusions:The result of this study showed that recombinant ROP1 Toxoplasma was produced successfully. In the present study, unlike other studies, the goal was to express full length of ROP1. Also this protein was produced alone not fused with other proteins.

Epidemiologic evaluation of toxoplasmosis and leading risk factors in HIV/AIDS patients in Arak City, Iran

“…Genomic DNA was extracted using phenolchloroform extraction protocol. 16,17 Standard primers were used for polymerase chain reaction (PCR), as follows: 18 forward, ‫׳5‬ -CGCTGCAGGGAGGAAGACGAAAGTTG-3‫׳‬and reverse, ‫-׳5‬ CGCTGCAGACACAGTGCATCTGGATT-3‫.׳‬ Optimization of the PCR reaction was carried out with a Master-Mix kit (CinnaGen Co.), and amplification was performed in a final volume of 25µl on an Eppendorf thermocycler with 5 min incubation at 94˚C, followed by 35 cycles of 30 min at 94˚C, 30 min at 58˚C, 30 min at 72˚C, and a final 10-min incubation at 72˚C. The amplified product was analysed using electrophoresis on a 1 per cent agarose gel.…”

Construction and Sequencing of Dense Granular14 (GRA14) Gene of Toxoplasma gondii (RH) in Expression Prokaryotic Plasmid PET32a: A Preliminary Study in Vaccine Production

“…Re combination is an easy method to access this specific protein. 30,31 In recent years, many progresses have been achieved in identifying candidate antigens of vaccine which are capable to inducing protective immune response. A number of these antigens were expressed in prokaryotic organisms and after purifying used in diagnostic studies.…”

Isolation and Genotyping of Acanthamoeba from Soil Samples in Markazi Province, Iran

“…According to the literature, the phenol-chloroform method was used for DNA extraction [12, 13]. For PCR amplification, genus-specific primers were used.…”