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

Introduction: 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 dense granule protein 14 (GRA14) gene is one of the excretory-secretary antigens of Toxoplasma which seems to be an appropriate candidate in production of recombinant vaccines and diagnostic kits. The current study aimed to clone GRA14 gene of T. gondii (RH) in a cloning vector for further production of dense granular proteins. Materials and Methods: Genomic DNA was isolated from tachyzoite of parasite by phenol chloroform method and gene fragment was amplified by polymerase chain reaction (PCR). The PCR products were ligated into restriction enzymes sites of pTG19-T cloning vector. Then transformed into Escherichia coli Top10 strain and screened by IPTG and X-Gal. Then recombinant plasmid confirmed by the colony-PCR and restriction enzyme digestion using SacI and NotI was done followed by sequencing .After isolation of this gene from pTG19-T, it was subcloned into a prokaryotic expression plasmid (pET32a). The pET32a - GRA14 constructs were analyzed by PCR, restriction analysis and sequencing. Results: Evaluation of PCR products by agarose gel electrophoresis and analysis of nucleotide sequencing of 1227 bp gene encoding the protein GRA14, revealed the complete homology with the recorded sequences in the gene bank. After enzyme restriction and electrophoresis a fragment about 1227 bp was separated from pET32a. Conclusions: The result of this study showed that recombinant GRA14 Toxoplasma was constructed successfully and ready for future study which seems like the antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit.

Keywords:

Toxoplasma gondii , Dense Granule Antigen 14 , Cloning , Subcloning